Beta2-adrenergic receptor agonists

ABSTRACT

Disclosed are multibinding compounds which are β2 adrenergic receptor agonists and are useful in the treatment and prevention of respiratory diseases such as asthma, bronchitis. They are also useful in the treatment of nervous system injury and premature labor.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/323,943, filed on Jun. 2, 1999 which claims the benefit ofU.S. patent application Ser. No. 60/088,466, filed Jun. 8, 1998; andU.S. patent application Ser. No. 60/092,938, filed Jul. 15, 1998; thedisclosures of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to novel multibinding compounds (agents)that are β2 adrenergic receptor agonists, partial agonists andpharmaceutical compositions comprising such compounds. Accordingly, themultibinding compounds and pharmaceutical compositions of this inventionare useful in the treatment and prevention of respiratory diseases suchas asthma, chronic obstructive pulmonary disease and chronic bronchitis.They are also useful in the treatment of nervous system injury andpremature labor.

[0004] 2. References

[0005] The following publications are cited in this application assuperscript numbers:

[0006]¹Hardman, J. G., et al. “The Pharmacological Basis ofTherapeutics”, McGraw-Hill, N.Y., (1996)

[0007]²Strosberg, A. D. “Structure, Function, and Regulation ofAdrenergic Receptors” Protein Sci. 2, 1198-1209 (1993).

[0008]³Beck-Sickinger, A. G. “Structure Characterization and BindingSites of G-Protein-coupled Receptors” DDT, 1, 502-513, (1996).

[0009]⁴Hein, L. & Kobilka, B. K. “Adrenergic Receptor SignalTransduction and Regulation” Neuropharmacol, 34, 357-366, (1995).

[0010]⁵Strosberg, A. D. & Pietri-Rouxel, F. “Function, and Regulation ofβ3-Adrenoceptor” TiPS, 17, 373-381, (1996).

[0011]⁶Bames, P. J. “Current Therapies for Asthma” CHEST, 111:17S-26S,(1997).

[0012]⁷Jack, D. A. “A way of Looking at Agonism and Antagonism: Lessonsfrom Salbutamol, Salmeterol and other β-Adrenoceptor Agonists” Br. J.Clin. Pharmac. 31, 501-514, (1991).

[0013]⁸Kissei Pharmaceutical Co. Ltd.“2-Amino-1-(4-hydroxy-2-methyl-phenyl)propanol derivatives” JP-10152460(Publication date Jun. 9, 1998).

[0014] All of the above publications are herein incorporated byreference in their entirety to the sane extent as if each individualpublication was specifically and individually indicated to beincorporated by reference in its entirety.

[0015] 3. State of the Art

[0016] A receptor is a biological structure with one or more bindingdomains that reversibly complexes with one or more ligands, where thatcomplexation has biological consequences. Receptors can exist entirelyoutside the cell (extracellular receptors), within the cell membrane(but presenting sections of the receptor to the extracellular milieu andcytosol), or entirely within the cell (intracellular receptors). Theymay also function independently of a cell (e.g., clot formation).Receptors within the cell membrane allow a cell to communicate with thespace outside of its boundaries (i.e., signaling) as well as to functionin the transport of molecules and ions into and out of the cell.

[0017] A ligand is a binding partner for a specific receptor or familyof receptors. A ligand may be the endogenous ligand for the receptor oralternatively may be a synthetic ligand for the receptor such as a drug,a drug candidate or a pharmacological tool.

[0018] The super family of seven transmembrane proteins (7-TMs), alsocalled G-protein coupled receptors (GPCRs), represents one of the mostsignificant classes of membrane bound receptors that communicate changesthat occur outside of the cell's boundaries to its interior, triggeringa cellular response when appropriate. The G-proteins, when activated,affect a wide range of downstream effector systems both positively andnegatively (e.g., ion channels, protein kinase cascades, transcription,transmigration of adhesion proteins, and the like).

[0019] Adrenergic receptors (AR) are members of the G-protein coupledreceptors that are composed of a family of three receptor sub-types: α1(_(A,B,D))α2 (_(A,B,C)), and β(_(1,2,3)).¹⁻⁵ These receptors areexpressed in tissues of various systems and organs of mammals and theproportions of the α and the β receptors are tissue dependant. Forexample, tissues of bronchial smooth muscle express largely β2-AR whilethose of cutaneous blood vessels contain exclusively α-AR subtypes.

[0020] It has been established that the β2-AR sub-type is involved inrespiratory diseases such as such as asthma⁶, chronic bronchitis,nervous system injury, and premature labor⁸. Currently, a number ofdrugs e.g., albuterol, formoterol, isoprenolol, or salmeterol havingβ2-AR agonist activities are being used to treat asthma However, thesedrugs have limited utility as they are either non-selective therebycausing adverse side effects such as muscle tremor, tachycardia,palpitations, and restlesness⁶, or have short duration of action and/orslow onset time of action.⁷ Accordingly, there is a need forβ2-selective AR agonists that are fast acting and have increased potencyand/or longer duration of action.

[0021] The multibinding compounds of the present invention fulfill thisneed.

SUMMARY OF THE INVENTION

[0022] This invention is directed to novel multibinding compounds(agents) that are agonists or partial agonists of β2 adrenergic receptorand are therefore useful in the treatment and prevention of respiratorydiseases such as asthma, chronic obstructive pulmonary disease, andchronic bronchitis. They are also useful in the treatment of nervoussystem injury and premature labor.

[0023] Accordingly, in one of its composition aspects, this inventionprovides a multibinding compound of Formula (I):

(L)_(p)(X)_(q)  (I)

[0024] wherein:

[0025] p is an integer of from 2 to 10;

[0026] q is an integer of from 1 to 20;

[0027] X is a linker; and

[0028] L is a ligand wherein:

[0029] one of the ligands, L, is a compound of formula (a):

[0030] wherein:

[0031] Ar¹ and Ar² are independently selected from the group consistingof aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, andheterocyclyl wherein each of said Ar¹ and Ar² substituent optionallylinks the ligand to a linker;

[0032] R¹ is selected from the group consisting of hydrogen, alkyl, andsubstituted alkyl, or R¹ is a covalent bond linking the ligand to alinker;

[0033] R² is selected from the group consisting of hydrogen, alkyl,aralkyl, acyl, substituted alkyl, cycloalkyl, and substitutedcycloalkyl, or R² is a covalent bond linking the ligand to a linker;

[0034] W is a covalent bond linking the —NR²— group to Ar², alkylene orsubstituted alkylene wherein one or more of the carbon atoms in saidalkylene or substituted alkylene group is optionally replaced by asubstituent selected from the group consisting of —NR^(a)— (where R^(a)is hydrogen, alkyl, acyl, or a covalent bond linking the ligand to alinker), —O—, —S(O)_(n) (where n is an integer of from 0 to 2), —CO—,—PR^(b)— (where R^(b) is alkyl), —P(O)₂—, and —O—P(O)O— and furtherwherein said alkylene or substituted alkylene group optionally links theligand to a linker provided that at least one of Ar¹, Ar², R¹, R², or Wlinks the ligand to a linker; and

[0035] the other ligands are independently of each other a compound offormula (b):

—Q—Ar³  (b)

[0036] wherein:

[0037] Ar³ is selected from the group consisting of aryl, heteroaryl,cycloalkyl, substituted cycloalkyl, and heterocyclyl;

[0038] Q, which links the other ligand to the linker, is selected fromthe group consisting of a covalent bond, alkylene, and substitutedalkylene wherein one or more of the carbon atoms in said alkylene andsubstituted alkylene is optionally replaced by a substituent selectedfrom the group consisting of —NR^(a)— (where R^(a) is hydrogen, alkyl,acyl, or a covalent bond linking the ligand to a linker), —O—,—S(O)_(n)— (where n is an integer of from 0 to 2), —CO—, —PR^(b)— (whereR^(b) is alkyl), —P(O)₂—, and —O—P(O)O—; and

[0039] individual isomers, mixtures of isomers and pharmaceuticallyacceptable salts thereof provided that:

[0040] (i) when the multibinding compound of Formula (I) is a compoundof formula:

[0041]  where Ar¹ and Ar³ are aryl, then W and X both are not alkyleneor alkylene —O—;

[0042] (ii) when the multibinding compound of Formula (I) is a compoundof formula:

[0043]  where Ar¹ is 4-hydroxy-2-methylphenyl, Ar² is aryl, Ar³ is arylor heterocyclyl, W is ethylene, Q is a covalent bond, R¹ is alkyl, thenthe linker X is not linked to the Ar² group through an oxygen atom;

[0044] (iii) when the multibinding compound of Formula (I) is a compoundof formula:

[0045]  where Ar¹ and Ar³ are aryl, W is alkylene, Ar² is aryl orcycloalkyl, Q is a covalent bond, then X is not -alkylene —O—; and

[0046] (iv) when the multibinding compound of Formula (I) is a compoundof formula:

[0047]  where Ar¹ is 4-benzyloxy-3-formylamino, R² is aralkyl, W is—CH(CH₃)CH₂—, Ar² and A³ are phenyl, Q is a covalent bond, then thelinker X is not linked to the Ar² group through an oxygen atom.

[0048] More preferably, each linker, X, in the multibinding compound ofFormula (I) independently has the formula:

—X^(a)—Z—(Y^(a)—Z)_(m)—X^(a)—

[0049] wherein

[0050] m is an integer of from 0 to 20;

[0051] X^(a) at each separate occurrence is selected from the groupconsisting of —O—, —S—, —NR—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)NR—,—NRC(O)—, C(S), —C(S)O—, —C(S)NR—, —NRC(S)—, or a covalent bond where Ris as defined below;

[0052] Z at each separate occurrence is selected from the groupconsisting of alkylene, substituted alkylene, cycloalkylene, substitutedcycloalkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, cycloalkenylene, substituted cycloalkenylene,arylene, heteroarylene, heterocyclene, or a covalent bond;

[0053] each Y^(a) at each separate occurrence is selected from the groupconsisting of —O—, —C(O)—, —OC(O)—, —C(O)O—, —NR—, —S(O)_(n)—,—C(O)NR′—, —NR′C(O)—, —NR′C(O)NR′—, —NR′C(S)NR′—, —C(═NR′)—NR′,—NR′—C(═NR′)—, —OC(O)—NR′—, —NR′—C(O)—O—, —N═C(X^(a))—NR′—,—NR′—C(X^(a))═N—, —P(O)(OR′)—O—, —O—P(O)(OR′)—, —S(O)_(n)CR′R″—,—S(O)_(n)—NR′—S(O)_(n)—, —S—S—, and a covalent bond; where n is 0, 1 or2; R, R′ and R″ at each separate occurrence are selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl,substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryland heterocyclic, and X^(a) is as defined above.

[0054] Preferably, q is less than p in the multibinding compounds ofthis invention.

[0055] In still another of its composition aspects, this inventionprovides a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and an effective amount of a multibinding compound ofFormula (I):

(L)_(p)(X)_(q)  (I)

[0056] wherein:

[0057] p is an integer of from 2 to 10;

[0058] q is an integer of from 1 to 20;

[0059] X is a linker;and

[0060] L is a ligand wherein:

[0061] one of the ligands, L, is a compound of formula (a):

[0062] wherein:

[0063] Ar₁ and Ar² are independently selected from the group consistingof aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, andheterocyclyl wherein each of said Ar¹ and Ar² substituent optionallylinks the ligand to a linker;

[0064] R¹ is selected from the group consisting of hydrogen, alkyl, andsubstituted alkyl, or R¹ is a covalent bond linking the ligand to alinker;

[0065] R² is selected from the group consisting of hydrogen, alkyl,aralkyl, acyl, substituted alkyl, cycloalkyl, and substitutedcycloalkyl, or R² is a covalent bond linking the ligand to a linker;

[0066] W is a covalent bond linking the —NR²— group to Ar², alkylene orsubstituted alkylene wherein one or more of the carbon atoms in saidalkylene and substituted alkylene is optionally replaced by asubstituent selected from the group consisting of —NR^(a)— (where R^(a)is hydrogen, alkyl, acyl, or a covalent bond linking the ligand to alinker), —O—, —S(O)_(n) (where n is an integer of from 0 to 2), —CO—,—PR^(b)— (where R^(b) is alkyl), —P(O)²—, and —O—P(O)O— and furtherwherein said alkylene or substituted alkylene group optionally links theligand to a linker provided that at least one of Ar¹, Ar², R¹, R², or Wlinks the ligand to a linker; and

[0067] the other ligands are independently of each other a compound offormula (b):

—Q—Ar³  (b)

[0068] wherein:

[0069] Ar³ is selected from the group consisting of aryl, heteroaryl,cycloalkyl, substituted cycloalkyl, and heterocyclyl;

[0070] Q, which links the other ligand to the linker, is selected fromthe group consisting of a covalent bond, alkylene, or a substitutedalkylene group wherein one or more of the carbon atoms in said alkyleneor substituted alkylene group is optionally replaced by a substituentselected from the group consisting of —NR^(a)— (where R^(a) is hydrogen,alkyl, acyl, or a covalent bond linking the ligand to a linker), —O—,—S(O)_(n)— (where n is an integer of from 0 to 2), —CO—, —PR^(b)— (whereR^(b) is alkyl), —P(O)₂—, and —O—P(O)O—; and individual isomers,mixtures of isomers and pharmaceutically acceptable salts thereofprovided that:

[0071] (i) when the multibinding compound of Formula (I) is a compoundof formula:

[0072]  where Ar¹ and Ar³ are aryl, then W and X both are not alkyleneor alkylene —O—;

[0073] (ii) when the multibinding compound of Formula (I) is a compoundof formula:

[0074]  where Ar¹ is 4-hydroxy-2-methylphenyl, Ar² is aryl, Ar³ is arylor heterocyclyl, W is ethylene, Q is a covalent bond, R¹ is alkyl, thenthe linker X is not linked to the Ar² group through an oxygen atom;

[0075] (iii) when the multibinding compound of Formula (I) is a compoundof formula:

[0076]  where Ar¹, Ar², Ar³, R¹, R² are as defined above, W is alkylene,and Q is a covalent bond, then X is not -alkylene-O—; and

[0077] (iv) when the multibinding compound of Formula (I) is a compoundof formula:

[0078]  where Ar¹ is 4-benzyloxy-3-formylamino, R² is aralkyl, W is—CH(CH₃)CH₂—, Ar² and Ar³ are phenyl, Q is a covalent bond, then thelinker X is not linked to the Ar² group through an oxygen atom.

[0079] More preferably, each linker, X, in the multibinding compound ofFormula (I) independently has the formula:

—X^(a)—Z—(Y^(a)—Z)_(m)—X^(a)—

[0080] wherein

[0081] m is an integer of from 0 to 20;

[0082] X^(a) at each separate occurrence is selected from the groupconsisting of —O—, —S—, —NR—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)NR—,—NRC(O)—, C(S), —C(S)O—, —C(S)NR—, —NRC(S)—, or a covalent bond where Ris as defined below;

[0083] Z at each separate occurrence is selected from the groupconsisting of alkylene, substituted alkylene, cycloalkylene, substitutedcycloalkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, cycloalkenylene, substituted cycloalkenylene,arylene, heteroarylene, heterocyclene, or a covalent bond;

[0084] each Y^(a) at each separate occurrence is selected from the groupconsisting of —O—, —C(O)—, —OC(O)—, —C(O)O—, —NR—, —S(O)_(n)—,—C(O)NR′—, —NR′C(O)—, —NR′C(O)NR′—, —NR′C(S)NR′—, —C(═NR′)—NR′—,—NR′—C(═NR′)—, —OC(O)—NR′—, —NR′—C(O)—O—, —N═C(X^(a))—NR′—,—NR′—C(X^(a))═N—, —P(O)(OR′)—O—, —O—P(O)(OR′)—, —S(O)_(n)CR′R″—,—S(O)_(n)—NR′, —NR′—S(O)_(n)—, —S—S—, and a covalent bond; where n is 0,1 or 2; R, R′ and R″ at each separate occurrence are selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl,substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryland heterocyclic, and X^(a) is as defined above.

[0085] In still another aspect, this invention provides a method oftreating diseases mediated by a β2 adrenergic receptor in a mammal, saidmethod comprising administering to said mammal a therapeuticallyeffective amount of a pharmaceutical composition comprising apharmaceutically acceptable carrier and a multibinding compound ofFormula (I):

(L)_(p)(X)_(q)  (I)

[0086] wherein:

[0087] p is an integer of from 2 to 10;

[0088] q is an integer of from 1 to 20;

[0089] X is a linker; and

[0090] L is a ligand wherein:

[0091] one of the ligands, L, is a compound of formula (a):

[0092] wherein:

[0093] Ar¹ and Ar² are independently selected from the group consistingof aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, andheterocyclyl wherein each of said Ar¹ and Ar² substituent optionallylinks the ligand to a linker;

[0094] R¹ is selected from the group consisting of hydrogen, alkyl, andsubstituted alkyl, or R¹ is a covalent bond linking the ligand to alinker;

[0095] R² is selected from the group consisting of hydrogen, alkyl,aralkyl, acyl, substituted alkyl, cycloalkyl, and substitutedcycloalkyl, or R² is a covalent bond linking the ligand to a linker;

[0096] W is a covalent bond linking the —NR²— group to Ar², alkylene orsubstituted alkylene wherein one or more of the carbon atoms in saidalkylene and substituted alkylene group is optionally replaced by asubstituent selected from the group consisting of —NR^(a)— (where R^(a)is hydrogen, alkyl, acyl, or a covalent bond linking the ligand to alinker), —O—, —S(O)_(n) (where n is an integer of from 0 to 2), —CO—,—PR^(b)— (where R^(b) is alkyl), —P(O)₂, and —O—P(O)O— and furtherwherein said alkylene or substituted alkylene group optionally links theligand to a linker provided that at least one of Ar¹, Ar², R¹, R², or Wlinks the ligand to a linker; and

[0097] the other ligands are independently of each other a compound offormula (b):

—Q—Ar³  (b)

[0098] wherein:

[0099] Ar³ is selected from the group consisting of aryl, heteroaryl,cycloalkyl, substituted cycloalkyl, and heterocyclyl;

[0100] Q, which links the other ligand to the linker, is selected fromthe group consisting of a covalent bond, alkylene, and substitutedalkylene wherein one or more of the carbon atoms in said alkylene andsubstituted alkylene group is optionally replaced by a substituentselected from the group consisting of —NR^(a)— (where R^(a) is hydrogen,alkyl, acyl, or a covalent bond linking the ligand to a linker), —O—,—S(O)_(n)— (where n is an integer of from 0 to 2), —CO—, —PR^(b)— (whereR^(b) is alkyl), —P(O)₂—, and —O—P(O)O—; and individual isomers,mixtures of isomers and pharmaceutically acceptable salts thereofprovided that:

[0101] (i) when the multibinding compound of Formula (I) is a compoundof formula:

[0102]  where Ar¹ and Ar³ are aryl, then W and X both are not alkyleneor alkylene-O—;

[0103] (ii) when the multibinding compound of Formula (I) is a compoundof formula:

[0104]  where Ar¹ is 4-hydroxy-2-methylphenyl, Ar² is aryl, Ar³ is arylor heterocyclyl, W is ethylene, Q is a covalent bond, R¹ is alkyl, thenthe linker X is not linked to the A² group through an oxygen atom;

[0105] (iii) when the multibinding compound of Formula (I) is a compoundof formula:

[0106]  where Ar¹, Ar², Ar³, R¹, R² are as defined above, W is alkylene,and Q is a covalent bond, then X is not -alkylene-O—; and

[0107] (iv) when the multibinding compound of Formula (I) is a compoundof formula:

[0108]  where Ar¹ is 4-benzyloxy-3-formylamino, R² is aralkyl, W is—CH(CH₃)CH₂—, Ar² and Ar³ are phenyl, Q is a covalent bond, then thelinker X is not linked to the Ar² group through an oxygen atom.

[0109] More preferably, each linker, X, in the multibinding compound ofFormula (I) independently has the formula:

—X^(a)—Z—(Y^(a)—Z)_(m)—X^(a)—

[0110] wherein

[0111] m is an integer of from 0 to 20;

[0112] X^(a) at each separate occurrence is selected from the groupconsisting of —O—, —S—, —N—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)NR—,—NRC(O)—, C(S), —C(S)O—, —C(S)NR—, —NRC(S)—, or a covalent bond where Ris as defined below;

[0113] Z at each separate occurrence is selected from the groupconsisting of alkylene, substituted alkylene, cycloalkylene, substitutedcycloalkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, cycloalkenylene, substituted cycloalkenylene,arylene, heteroarylene, heterocyclene, or a covalent bond;

[0114] each Y^(a) at each separate occurrence is selected from the groupconsisting of —O—, —C(O)—, —OC(O)—, —C(O)O—, —NR—, —S(O)_(n)—,—C(O)NR′—, —NR′C(O)—, —NR′C(O)NR′—, —NR′C(S)NR′—, —C(═NR′)—NR′—,—NR′—C(═NR′)—, —OC(O)—NR′—, —NR′—C(O)—O—, —N═C(X^(a))—NR′—,—NR′—C(X^(a))═N—,—P(O)(OR′)—O—, —O—P(O)(OR′)—, —S(O)_(n)CR′R″—,—S(O)_(n)—NR′, —NR′—S(O)_(n)—, —S—S—, and a covalent bond; where n is 0,1 or 2; R, R′ and R″ at each separate occurrence are selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl,substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryland heterocyclic, and X^(a) is as defined above.

[0115] Preferably p is less than q.

[0116] In still another aspect, this invention is directed to generalsynthetic methods for generating large libraries of diverse multimericcompounds which multimeric compounds are candidates for possessingmultibinding properties for β2 adrenergic receptor. The diversemultimeric compound libraries provided by this invention are synthesizedby combining a linker or linkers with a ligand or ligands to provide fora library of multimeric compounds wherein the linker and ligand eachhave complementary functional groups permitting covalent linkage. Thelibrary of linkers is preferably selected to have diverse propertiessuch as valency, linker length, linker geometry and rigidity,hydrophilicity or hydrophobicity, amphiphilicity, acidity, basicity andpolarization. The library of ligands is preferably selected to havediverse attachment points on the same ligand, different functionalgroups at the same site of otherwise the same ligand, and the like.

[0117] This invention is also directed to libraries of diversemultimeric compounds which multimeric compounds are candidates forpossessing multibinding properties for β2 adrenergic receptor. Theselibraries are prepared via the methods described above and permit therapid and efficient evaluation of what molecular constraints impartmultibinding properties to a ligand or a class of ligands targeting areceptor.

[0118] Accordingly, in one of its method aspects, this invention isdirected to a method for identifying multimeric ligand compoundspossessing multibinding properties for β2 adrenergic receptor whichmethod comprises:

[0119] (a) identifying a ligand or a mixture of ligands wherein eachligand contains at least one reactive functionality;

[0120] (b) identifing a library of linkers wherein each linker in saidlibrary comprises at least two functional groups having complementaryreactivity to at least one of the reactive functional groups of theligand;

[0121] (c) preparing a multimeric ligand compound library by combiningat least two stoichiometric equivalents of the ligand or mixture ofligands identified in (a) with the library of linkers identified in (b)under conditions wherein the complementary functional groups react toform a covalent linkage between said linker and at least two of saidligands; and

[0122] (d) assaying the multimeric ligand compounds produced in (c)above to identify multimeric ligand compounds possessing multibindingproperties for β2 adrenergic receptor.

[0123] In another of its method aspects, this invention is directed to amethod for identifying multimeric ligand compounds possessingmultibinding properties for β2 adrenergic receptor which methodcomprises:

[0124] (a) identifying a library of ligands wherein each ligand containsat least one reactive functionality;

[0125] (b) identifying a linker or mixture of linkers wherein eachlinker comprises at least two functional groups having complementaryreactivity to at least one of the reactive functional groups of theligand;

[0126] (c) preparing a multimeric ligand compound library by combiningat least two stoichiometric equivalents of the library of ligandsidentified in (a) with the linker or mixture of linkers identified in(b) under conditions wherein the complementary functional groups reactto form a covalent linkage between said linker and at least two of saidligands; and

[0127] (d) assaying the multimeric ligand compounds produced in (c)above to identify multimeric ligand compounds possessing multibindingproperties for β2 adrenergic receptor.

[0128] The preparation of the multimeric ligand compound library isachieved by either the sequential or concurrent combination of the twoor more stoichiometric equivalents of the ligands identified in (a) withthe linkers identified in (b). Sequential addition is preferred when amixture of different ligands is employed to ensure heterodimeric ormultimeric compounds are prepared. Concurrent addition of the ligandsoccurs when at least a portion of the multimer comounds prepared arehomomultimeric compounds.

[0129] The assay protocols recited in (d) can be conducted on themultimeric ligand compound library produced in (c) above, or preferably,each member of the library is isolated by preparative liquidchromatography mass spectrometry (LCMS).

[0130] In one of its composition aspects, this invention is directed toa library of multimeric ligand compounds which may possess multivalentproperties for β2 adrenergic receptor which library is prepared by themethod comprising:

[0131] (a) identifying a ligand or a mixture of ligands wherein eachligand contains at least one reactive functionality;

[0132] (b) identifying a library of linkers wherein each linker in saidlibrary comprises at least two functional groups having complementaryreactivity to at least one of the reactive functional groups of theligand; and

[0133] (c) preparing a multimeric ligand compound library by combiningat least two stoichiometric equivalents of the ligand or mixture ofligands identified in (a) with the library of linkers identified in (b)under conditions wherein the complementary functional groups react toform a covalent linkage between said linker and at least two of saidligands.

[0134] In another of its composition aspects, this invention is directedto a library of multimeric ligand compounds which may possessmultivalent properties for β2 adrenergic receptor which library isprepared by the method comprising:

[0135] (a) identifying a library of ligands wherein each ligand containsat least one reactive functionality;

[0136] (b) identifying a linker or mixture of linkers wherein eachlinker comprises at least two functional groups having complementaryreactivity to at least one of the reactive functional groups of theligand; and

[0137] (c) preparing a multimeric ligand compound library by combiningat least two stoichiometric equivalents of the library of ligandsidentified in (a) with the linker or mixture of linkers identified in(b) under conditions wherein the complementary functional groups reactto form a covalent linkage between said linker and at least two of saidligands.

[0138] In a preferred embodiment, the library of linkers employed ineither the methods or the library aspects of this invention is selectedfrom the group comprising flexible linkers, rigid linkers, hydrophobiclinkers, hydrophilic linkers, linkers of different geometry, acidiclinkers, basic linkers, linkers of different polarization andamphiphilic linkers. For example, in one embodiment, each of the linkersin the linker library may comprise linkers of different chain lengthand/or having different complementary reactive groups. Such linkerlengths can preferably range from about 2 to 100 Å.

[0139] In another preferred embodiment, the ligand or mixture of ligandsis selected to have reactive functionality at different sites on saidligands in order to provide for a range of orientations of said ligandon said multimeric ligand compounds. Such reactive functionalityincludes, by way of example, carboxylic acids, carboxylic acid halides,carboxyl esters, amines, halides, isocyanates, vinyl unsaturation,ketones, aldehydes, thiols, alcohols, anhydrides, and precursorsthereof. It is understood, of course, that the reactive functionality onthe ligand is selected to be complementary to at least one of thereactive groups on the linker so that a covalent linkage can be formedbetween the linker and the ligand.

[0140] In other embodiments, the multimeric ligand compound is homomeric(i.e., each of the ligands is the same, although it may be attached atdifferent points) or heterodimeric (i.e., at least one of the ligands isdifferent from the other ligands).

[0141] In addition to the combinatorial methods described herein, thisinvention provides for an interative process for rationally evaluatingwhat molecular constraints impart multibinding properties to a class ofmultimeric compounds or ligands targeting a receptor. Specifically, thismethod aspect is directed to a method for identifying multimeric ligandcompounds possessing multibinding properties for β2 adrenergic receptorwhich method comprises:

[0142] (a) preparing a first collection or iteration of multimericcompounds which is prepared by contacting at least two stoichiometricequivalents of the ligand or mixture of ligands which target a receptorwith a linker or mixture of linkers wherein said ligand or mixture ofligands comprises at least one reactive functionality and said linker ormixture of linkers comprises at least two functional groups havingcomplementary reactivity to at least one of the reactive functionalgroups of the ligand wherein said contacting is conducted underconditions wherein the complementary functional groups react to form acovalent linkage between said linker and at least two of said ligands;

[0143] (b) assaying said first collection or iteration of multimericcompounds to assess which if any of said multimeric compounds possessmultibinding properties for β2 adrenergic receptor;

[0144] (c) repeating the process of (a) and (b) above until at least onemultimeric compound is found to possess multibinding properties for β2adrenergic receptor;

[0145] (d) evaluating what molecular constraints imparted multibindingproperties for β2 adrenergic receptor to the multimeric compound orcompounds found in the first iteration recited in (a)-(c) above;

[0146] (e) creating a second collection or iteration of multimericcompounds which elaborates upon the particular molecular constraintsimparting multibinding properties to the multimeric compound orcompounds found in said first iteration;

[0147] (f) evaluating what molecular constraints imparted enhancedmultibinding properties to the multimeric compound or compounds found inthe second collection or iteration recited in (e) above;

[0148] (g) optionally repeating steps (e) and (f) to further elaborateupon said molecular constraints.

[0149] Preferably, steps (e) and (f) are repeated at least two times,more preferably at from 2-50 times, even more preferably from 3 to 50times, and still more preferably at least 5-50 times.

BRIEF DESCRIPTION OF THE DRAWINGS

[0150]FIG. 1 illustrates examples of multibinding compounds comprising 2ligands attached in different formats to a linker.

[0151]FIG. 2 illustrates examples of multibinding compounds comprising 3ligands attached in different formats to a linker.

[0152]FIG. 3 illustrates examples of multibinding compounds comprising 4ligands attached in different formats to a linker.

[0153]FIG. 4 illustrates examples of multibinding compoundscomprising >4 ligands attached in different formats to a linker.

[0154] FIGS. 5-15 illustrate synthesis of compounds of Formula (I).

DETAILED DESCRIPTION OF THE INVENTION Definitions

[0155] This invention is directed to multibinding compounds which are β2adrenergic receptor agonists pharmaceutical compositions containing suchcompounds and methods for treating diseases mediated by β2 adrenergicreceptor in mammals. When discussing such compounds, compositions ormethods, the following terms have the following meanings unlessotherwise indicated. Any undefined terms have their art recognizedmeanings.

[0156] The term “alkyl” refers to a monoradical branched or unbranchedsaturated hydrocarbon chain preferably having from 1 to 40 carbon atoms,more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6carbon atoms. This term is exemplified by groups such as methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, n-decyl, tetradecyl,and the like.

[0157] The term “substituted alkyl” refers to an alkyl group as definedabove, having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl. This term is exemplified by groups such ashydroxymethyl, hydroxyethyl, hydroxypropyl, 2-aminoethyl, 3-aminopropyl,2-methylaminoethyl, 3-dimethylaminopropyl, 2-sulfonamidoethyl,2-carboxyethyl, and the like.

[0158] The term “alkylene” refers to a diradical of a branched orunbranched saturated hydrocarbon chain, preferably having from 1 to 40carbon atoms, more preferably 1 to 10 carbon atoms and even morepreferably 1 to 6 carbon atoms. This term is exemplified by groups suchas methylene (—CH₂—), ethylene (—CH₂CH₂—), the propylene isomers (e.g.,—CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like.

[0159] The term “substituted alkylene” refers to an alkylene group, asdefined above, having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substitutedamino,-aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen,hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl. Additionally,such substituted alkylene groups include those where 2 substituents onthe alkylene group are fused to form one or more cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heterocyclicor heteroaryl groups fused to the alkylene group. Preferably such fusedgroups contain from 1 to 3 fused ring structures.

[0160] The term “alkaryl” or “aralkyl” refers to the groups-alkylene-aryl and -substituted alkylene-aryl where alkylene,substituted alkylene and aryl are defined herein. Such alkaryl groupsare exemplified by benzyl, phenethyl and the like.

[0161] The term “heteroaralkyl” refers to the groups-alkylene-heteroaryl and -substituted alkylene-heteroaryl wherealkylene, substituted alkylene and heteroaryl are defined herein. Suchheteroaralkyl groups are exemplified by pyridin-3-lmethyl,pyridin-3-ylmethyloxy, and the like.

[0162] The term “alkoxy” refers to the groups alkyl-O—, alkenyl-O—,cycloalkyl-O—, cycloalkenyl-O—, and alkynyl-O—, where alkyl, alkenyl,cycloalkyl, cycloalkenyl, and alkynyl are as defined herein. Preferredalkoxy groups are alkyl-O— and include, by way of example, methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy,n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

[0163] The term “substituted alkoxy” refers to the groups substitutedalkyl-O—, substituted alkenyl-O—, substituted cycloalkyl-O—, substitutedcycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyland substituted alkynyl are as defined herein.

[0164] The term “alkenyl” refers to a monoradical of a branched orunbranched unsaturated hydrocarbon group preferably having from 2 to 40carbon atoms, more preferably 2 to 10 carbon atoms and even morepreferably 2 to 6 carbon atoms and having at least 1 and preferably from1-6 sites of vinyl unsaturation. Preferred alkenyl groups includeethenyl (—CH═CH₂), n-propenyl (—CH₂CH═CH₂), iso-propenyl (—C(CH₃)═CH₂),and the like.

[0165] The term “substituted alkenyl” refers to an alkenyl group asdefined above having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl.

[0166] The term “alkenylene” refers to a diradical of a branched orunbranched unsaturated hydrocarbon group preferably having from 2 to 40carbon atoms, more preferably 2 to 10 carbon atoms and even morepreferably 2 to 6 carbon atoms and having at least 1 and preferably from1-6 sites of vinyl unsaturation. This term is exemplified by groups suchas ethenylene (—CH═CH—), the propenylene isomers (e.g., —CH₂CH═CH—,—C(CH₃)═CH—, and the like.

[0167] The term “substituted alkenylene” refers to an alkenylene groupas defined above having from 1 to 5 substituents, and preferably from 1to 3 substituents, selected from the group consisting of alkoxy,substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substitutedamino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen,hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl. Additionally,such substituted alkenylene groups include those where 2 substituents onthe alkenylene group are fused to form one or more cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,heterocyclic or heteroaryl groups fused to the alkenylene group.

[0168] The term “alkynyl” refers to a monoradical of an unsaturatedhydrocarbon preferably having from 2 to 40 carbon atoms, more preferably2 to 20 carbon atoms and even more preferably 2 to 6 carbon atoms andhaving at least 1 and preferably from 1-6 sites of acetylene (triplebond) unsaturation. Preferred alkynyl groups include ethynyl (—C≡CH),propargyl (—CH₂C≡CH) and the like.

[0169] The term “substituted alkynyl” refers to an alkynyl group asdefined above having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl,and —SO₂-heteroaryl.

[0170] The term “alkynylene” refers to a diradical of an unsaturatedhydrocarbon preferably having from 2 to 40 carbon atoms, more preferably2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms andhaving at least 1 and preferably from 1-6 sites of acetylene (triplebond) unsaturation. Preferred alkynylene groups include ethynylene(—C≡C—), propargylene (—CH₂C≡C—) and the like.

[0171] The term “substituted alkynylene” refers to an alkynylene groupas defined above having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl

[0172] The term “acyl” refers to the groups HC(O)—, alkyl-C(O)—,substituted alkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—,cycloalkyl-C(O)—, substituted cycloalkyl-C(O)—, cycloalkenyl-C(O)—,substituted cycloalkenyl-C(O)—, aryl-C(O)—, heteroaryl-C(O)—andheterocyclic-C(O)—where alkyl, substituted alkyl, alkenyl, substitutedalkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, heteroaryl and heterocyclic are as defined herein.

[0173] The term “acylamino” or “aminocarbonyl” refers to the group—C(O)NRR where each R is independently hydrogen, alkyl, substitutedalkyl, aryl, heteroaryl, heterocyclic or where both R groups are joinedto form a heterocyclic group (e.g., morpholino) wherein alkyl,substituted alkyl, aryl, heteroaryl and heterocyclic are as definedherein.

[0174] The term “sulfonylamino” refers to the group —NRSO₂R^(a) where Ris hydrogen, alkyl, substituted alkyl, aralkyl, or heteroaralkyl, andR^(a) is alkyl, substituted alkyl, amino, or substituted amino whereinalkyl, substituted alkyl, aralkyl, heteroaralkyl and substituted aminoare as defined herein.

[0175] The term “aminoacyl” refers to the group —NRC(O)R where each R isindependently hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, amino, substituted amino, aryl, heteroaryl, or heterocyclicwherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl,heteroaryl and heterocyclic are as defined herein.

[0176] The term “aminoacyloxy” or “alkoxycarbonylamino” refers to thegroup —NRC(O)OR where each R is independently hydrogen, alkyl,substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl,substituted alkyl, aryl, heteroaryl and heterocyclic are as definedherein.

[0177] The term “acyloxy” refers to the groups alkyl-C(O)O—, substitutedalkyl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—,aryl-C(O)O—, heteroaryl-C(O)O—, and heterocyclic-C(O)O—wherein alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl,and heterocyclic are as defined herein.

[0178] The term “aryl” refers to an unsaturated aromatic carbocyclicgroup of from 6 to 20 carbon atoms having a single ring (e.g., phenyl)or multiple condensed (fused) rings (e.g., naphthyl or anthryl). Thearyl group may optionally be fused to a heterocyclic or cycloalkylgroup. Preferred aryls include phenyl, naphthyl and the like. Unlessotherwise constrained by the definition for the aryl substituent, sucharyl groups can optionally be substituted with from 1 to 5 substituents,preferably 1 to 3 substituents, selected from the group consisting ofacyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy,substituted alkenyl, substituted alkynyl, substituted cycloalkyl,substituted cycloalkenyl, amino, substituted amino, aminoacyl,acylamino, sulfonylamino, alkaryl, aryl, aryloxy, azido, carboxyl,carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy,heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl and trihalomethyl.Preferred aryl substituents include alkyl, alkoxy, halo, cyano, nitro,trihalomethyl, and thioalkoxy.

[0179] The term “aryloxy” refers to the group aryl-O—wherein the arylgroup is as defined above including optionally substituted aryl groupsas also defined above.

[0180] The term “arylene” refers to the diradical derived from aryl(including substituted aryl) as defined above and is exemplified by1,2-phenylene, 1,3-phenylene, 1,4-phenylene, 1,2-naphthylene and thelike.

[0181] The term “amino” refers to the group —NH₂.

[0182] The term “substituted amino” refers to the group —NRR where eachR is independently selected from the group consisting of hydrogen,alkyl, substituted alkyl, acyl, cycloalkyl, substituted cycloalkyl,alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl,alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic providedthat both R′s are not hydrogen.

[0183] The term “carboxyalkyl” or “alkoxycarbonyl” refers to the groups“—C(O)O-alkyl”, “—C(O)O-substituted alkyl”, “—C(O)O-cycloalkyl”,“—C(O)O-substituted cycloalkyl”, “—C(O)O-alkenyl”, “—C(O)O-substitutedalkenyl”, “—C(O)O-alkynyl” and “—C(O)O-substituted alkynyl” where alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, alkynyl and substituted alkynyl are as definedherein.

[0184] The term “cycloalkyl” refers to cyclic alkyl groups of from 3 to20 carbon atoms having a single cyclic ring or multiple condensed rings,said cycloalkyl group may optionally be fused to an aryl or heteroarylgroup. Such cycloalkyl groups include, by way of example, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, andthe like, or multiple ring structures such as adamantanyl, and the like.

[0185] The term “substituted cycloalkyl” refers to cycloalkyl groupshaving from 1 to 5 substituents, and preferably 1 to 3 substituents,selected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO²-aryl and —SO₂-heteroaryl.

[0186] The term “cycloalkenyl” refers to cyclic alkenyl groups of from 4to 20 carbon atoms having a single cyclic ring and at least one point ofinternal unsaturation. Examples of suitable cycloalkenyl groups include,for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and thelike.

[0187] The term “substituted cycloalkenyl” refers to cycloalkenyl groupshaving from 1 to 5 substituents, and preferably 1 to 3 substituents,selected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO²-aryl and —SO₂-heteroaryl.

[0188] The term “halo” or “halogen” refers to fluoro, chloro, bromo andiodo.

[0189] The term “heteroaryl” refers to an aromatic group of from 1 to 15carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen andsulfur within at least one ring (if there is more than one ring). Theheteroaryl ring may optionally be fused to a cycloalkyl or heterocyclylring. Unless otherwise constrained by the definition for the heteroarylsubstituent, such heteroaryl groups can be optionally substituted with 1to 5 substituents, preferably 1 to 3 substituents, selected from thegroup consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl,substituted alkoxy, substituted alkenyl, substituted allyl, substitutedcycloalkyl, substituted cycloalkenyl, amino, substituted amino,aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl,carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy,heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl and trihalomethyl.Preferred heteroaryl substituents include alkyl, alkoxy, halo, cyano,nitro, trihalomethyl, and thioalkoxy. Such heteroaryl groups can have asingle ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g.,indolizinyl or benzothienyl). Preferred heteroaryls include pyridyl,pyrrolyl and furyl.

[0190] The term “heteroaryloxy” refers to the group heteroaryl-O—.

[0191] The term “heteroarylene” refers to the diradical group derivedfrom heteroaryl (including substituted heteroaryl), as defined above,and is exemplified by the groups 2,6-pyridylene, 2,4-pyridinylene,1,2-quinolinylene, 1,8-quinolinylene, 1,4-benzofuranylene,2,5-pyridnylene, 2,5-indolenyl, and the like.

[0192] The term “cycloalkylene” refers to the diradical group derivedfrom cycloalkyl, as defined above, and is exemplified by the groups1,6-cyclohexylene, 1,3-cyclopentylene, and the like.

[0193] The term “substituted cycloalkylene” refers to the diradicalgroup derived from substituted cycloalkyl, as defined above.

[0194] The term “cycloalkenylene” refers to the diradical group derivedfrom cycloalkyl, as defined above.

[0195] The term “substituted cycloalkenylene” refers to the diradicalgroup derived from substituted cycloalkenyl, as defined above.

[0196] The term “heterocycle” or “heterocyclyl” refers to a monoradicalsaturated unsaturated group having a single ring or multiple condensedrings, from 1 to 40 carbon atoms and from 1 to 10 hetero atoms,preferably 1 to 4 heteroatoms, selected from nitrogen, sulfur,phosphorus, and/or oxygen within the ring and further wherein one, two,or three of the ring carbon atoms may optionally be replaced with acarbonyl group (i.e., a keto group). Unless otherwise constrained by thedefinition for the heterocyclic substituent, such heterocyclic groupscan be optionally substituted with 1 to 5, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl. Such heterocyclic groups can have a single ring ormultiple condensed rings. Preferred heterocyclics include morpholino,piperidinyl, and the like.

[0197] Examples of heteroaryls and heterocycles include, but are notlimited to, pyrrole, thiophene, furan, imidazole, pyrazole, pyridine,pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, pyrrolidine, piperidine, piperazine,indoline, morpholine, tetrahydrofuranyl, tetrahydrothiophene, and thelike as well as N-alkoxy-nitrogen containing heterocycles.

[0198] The term “heterocyclooxy” refers to the group heterocyclic-O—.

[0199] The term “thioheterocyclooxy” refers to the groupheterocyclic-S—.

[0200] The term “heterocyclene” refers to the diradical group formedfrom a heterocycle, as defined herein, and is exemplified by the groups2,6-morpholino, 2,5-morpholino and the like.

[0201] The term “oxyacylamino” or “aminocarbonyloxy” refers to the group—OC(O)NRR where each R is independently hydrogen, alkyl, substitutedalkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substitutedalkyl, aryl, heteroaryl and heterocyclic are as defined herein.

[0202] The term “spiro-attached cycloalkyl group” refers to a cycloalkylgroup joined to another ring via one carbon atom common to both rings.

[0203] The term “thiol” refers to the group —SH.

[0204] The term “thioalkoxy” or “alkylthio” refers to the group—S-alkyl.

[0205] The term “substituted thioalkoxy” refers to the group—S-substituted alkyl.

[0206] The term “thioaryloxy” refers to the group aryl-S— wherein thearyl group is as defined above including optionally substituted arylgroups also defined above.

[0207] The term “thioheteroaryloxy” refers to the group heteroaryl-S—wherein the heteroaryl group is as defined above including optionallysubstituted aryl groups as also defined above.

[0208] As to any of the above groups which contain one or moresubstituents, it is understood, of course, that such groups do notcontain any substitution or substitution patterns which are stericallyimpractical and/or synthetically non-feasible. In addition, thecompounds of this invention include all stereochemical isomers arisingfrom the substitution of these compounds.

[0209] The term “pharmaceutically-acceptable salts” refers to saltswhich retain the biological effectiveness and properties of themultibinding compounds of this invention and which are not biologicallyor otherwise undesirable. In many cases, the multibinding compounds ofthis invention are capable of forming acid and/or base salts by virtueof the presence of amino and/or carboxyl groups or groups similarthereto.

[0210] Pharmaceutically-acceptable base addition salts can be preparedfrom inorganic and organic bases. Salts derived from inorganic bases,include by way of example only, sodium, potassium, lithium, ammonium,calcium and magnesium salts. Salts derived from organic bases include,but are not limited to, salts of primary, secondary and tertiary amines,such as alkyl amines, dialkyl amines, trialkyl amines, substituted alkylamines, di(substituted alkyl) amines, tri(substituted alkyl) amines,alkenyl amines, dialkenyl amines, trialkenyl amines, substituted alkenylamines, di(substituted alkenyl) amines, tri(substituted alkenyl) amines,cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines,substituted cycloalkyl amines, disubstituted cycloalkyl amine,trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines,disubstituted cycloalkenyl amine, trisubstituted cycloalkenyl amines,aryl amines, diaryl amines, triaryl amines, heteroaryl amines,diheteroaryl amines, triheteroaryl amines, heterocyclic amines,diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amineswhere at least two of the substituents on the amine are different andare selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic,and the like. Also included are amines where the two or threesubstituents, together with the amino nitrogen, form a heterocyclic orheteroaryl group. Examples of suitable amines include, by way of exampleonly, isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl)amine, tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol,tromethamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,N-alkylglucamines, theobromine, purines, piperazine, piperidine,morpholine, N-ethylpiperidine, and the like. It should also beunderstood that other carboxylic acid derivatives would be useful in thepractice of this invention, for example, carboxylic acid amides,including carboxamides, lower alkyl carboxamides, dialkyl carboxamides,and the like.

[0211] Pharmaceutically acceptable acid addition salts may be preparedfrom inorganic and organic acids. Salts derived from inorganic acidsinclude hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, naphthoic acid, 2-hydroxynaphthoic acid, and the like.

[0212] The term “pharmaceutically-acceptable cation” refers to thecation of a pharmaceutically-acceptable salt.

[0213] The term “library” refers to at least 3, preferably from 10² to10⁹ and more preferably from 10² to 10⁴ multimeric compounds.Preferably, these compounds are prepared as a multiplicity of compoundsin a single solution or reaction mixture which permits facile synthesisthereof In one embodiment, the library of multimeric compounds can bedirectly assayed for multibinding properties. In another embodiment,each member of the library of multimeric compounds is first isolatedand, optionally, characterized. This member is then assayed formultibinding properties.

[0214] The term “collection” refers to a set of multimeric compoundswhich are prepared either sequentially or concurrently (e.g.,combinatorially). The collection comprises at least 2 members;preferably from 2 to 10⁹ members and still more preferably from 10 to10⁴ members.

[0215] The term “multimeric compound” refers to compounds comprisingfrom 2 to 10 ligands covalently connected through at least one linkerwhich compounds may or may not possess multibinding properties (asdefined herein).

[0216] The term “pseudohalide” refers to functional groups which reactin displacement reactions in a manner similar to a halogen. Suchfunctional groups include, by way of example, mesyl, tosyl, azido andcyano groups.

[0217] The term “protecting group” or “blocking group” refers to anygroup which when bound to one or more hydroxyl, thiol, amino or carboxylgroups of the compounds (including intermediates thereof) preventsreactions from occurring at these groups and which protecting group canbe removed by conventional chemical or enzymatic steps to reestablishthe hydroxyl, thiol, amino or carboxyl group (See., T. W. Greene and P.G. H. Wuts, “Protective Groups in Organic Synthesis”, 2^(nd) Ed.). Theparticular removable blocking group employed is not critical andpreferred removable hydroxyl blocking groups include conventionalsubstituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl,benzylidine, phenacyl, t-butyl-diphenylsilyl and any other group thatcan be introduced chemically onto a hydroxyl functionality and laterselectively removed either by chemical or enzymatic methods in mildconditions compatible with the nature of the product. Preferredremovable thiol blocking groups include disulfide groups, acyl groups,benzyl groups, and the like.

[0218] Preferred removable amino blocking groups include conventionalsubstituents such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ),fluorenylmethoxy-carbonyl (FMOC), allyloxycarbonyl (ALOC), and the likewhich can be removed by conventional conditions compatible with thenature of the product.

[0219] Preferred carboxyl protecting groups include esters such asmethyl, ethyl, propyl, t-butyl etc. which can be removed by mildconditions compatible with the nature of the product.

[0220] The term “optional” or “optionally” means that the subsequentlydescribed event, circumstance or substituent may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

[0221] The term “ligand” or “ligands” as used herein denotes a compoundthat is a binding partner for a β2 adrenergic receptor and is boundthereto by complementarity. Preferred ligands are those that are eitherβ2 adrenergic receptor agonists, partial agonists, or antagonists. Thespecific region or regions of the ligand that is (are) recognized by thereceptor is designated as the “ligand domain”. A ligand may be eithercapable of binding to the receptor by itself, or may require thepresence of one or more non-ligand components for binding (e.g., Ca⁺²,Mg⁺² or a water molecule is required for the binding of a ligand tovarious ligand binding sites). Examples of ligands useful in thisinvention are described herein. Those skilled in the art will appreciatethat portions of the ligand structure that are not essential forspecific molecular recognition and binding activity may be variedsubstantially, replaced or substituted with unrelated structures (forexample, with ancillary groups as defined below) and, in some cases,omitted entirely without affecting the binding interaction. The primaryrequirement for a ligand is that it has a ligand domain as definedabove. It is understood that the term ligand is not intended to belimited to compounds known to be useful in binding to β2 adrenergicreceptor (e.g., known drugs). Those skilled in the art will understandthat the term ligand can equally apply to a molecule that is notnormally associated with β2 adrenergic receptor binding properties. Inaddition, it should be noted that ligands that exhibit marginal activityor lack useful activity as monomers can be highly active as multivalentcompounds because of the benefits conferred by multivalency

[0222] The term “ligand” or “ligands” as used herein is intended toinclude the racemic forms of the ligands as well as individualenantiomers and diasteromers and non-racemic mixtures thereof.

[0223] The term “multibinding compound or agent” refers to a compoundthat is capable of multivalency, as defined below, and which has 2-10ligands covalently bound to one or more linkers. In all cases, eachligand and linker in the multibinding compound is independently selectedsuch that the multibinding compound includes both symmetric compounds(i.e., where each ligand as well as each linker is identical) andasymmetric compounds (i.e., where at least one of the ligands isdifferent from the other ligand(s) and/or at least one linker isdifferent from the other linker(s)). Multibinding compounds provide abiological and/or therapeutic effect greater than the aggregate ofunlinked ligands equivalent thereto which are made available forbinding. That is to say that the biological and/or therapeutic effect ofthe ligands attached to the multibinding compound is greater than thatachieved by the same amount of unlinked ligands made available forbinding to the ligand binding sites (receptors). The phrase “increasedbiological or therapeutic effect” includes, for example: increasedaffinity, increased selectivity for target, increased specificity fortarget, increased potency, increased efficacy, decreased toxicity,improved duration of activity or action, increased ability to kill cellssuch as fungal pathogens, cancer cells, etc., decreased side effects,increased therapeutic index, improved bioavailibity, improvedpharmacokinetics, improved activity spectrum, and the like. Themultibinding compounds of this invention will exhibit at least one andpreferably more than one of the above-mentioned affects.

[0224] The term “univalency” as used herein refers to a single bindinginteraction between one ligand as defined herein with one ligand bindingsite as defined herein. It should be noted that a compound havingmultiple copies of a ligand (or ligands) exhibit univalency when onlyone ligand is interacting with a ligand binding site. Examples ofunivalent interactions are depicted below.

[0225] The term “multivalency” as used herein refers to the concurrentbinding of from 2 to 10 linked ligands (which may be the same ordifferent) and two or more corresponding receptors (ligand bindingsites) which may be the same or different.

[0226] For example, two ligands connected through a linker that bindconcurrently to two ligand binding sites would be considered asbivalency; three ligands thus connected would be an example oftrivalency. An example of trivalent binding, illustrating a multibindingcompound bearing three ligands versus a monovalent binding interaction,is shown below:

[0227] It should be understood that not all compounds that containmultiple copies of a ligand attached to a linker or to linkersnecessarily exhibit the phenomena of multivalency, i.e., that thebiological and/or therapeutic effect of the multibinding agent isgreater than the sum of the aggregate of unlinked ligands made availablefor binding to the ligand binding site (receptor). For multivalency tooccur, the ligands that are connected by a linker or linkers have to bepresented to their ligand binding sites by the linker(s) in a specificmanner in order to bring about the desired ligand-orienting result, andthus produce a multibinding event.

[0228] Furthermore, the multibinding compound of the present inventioncan be composed of ligands that are all β2 adrenergic receptor agonists,partial agonists, or it can be composed of ligands that are selectedfrom β2 adrenergic receptor agonists and antagonists provided that themultibinding compounds exhibits an overall β2 adrenergic receptoragonistic or partial agonistic activity. A multibinding compound thatexhibits partial agonist activity at adrenoceptors may provideadvantages over a compound that exhibits full agonism. Partial agonismmay result in a reduction of the rate of receptor desensitization,receptor recycling, or receptor expression in mammalian tissue. This mayresult in increased therapeutic benefits from using such an agonistversus a compound which behaves as a full agonist for the chronictreatment of pathological conditions or diseases. A multibindingcompound may also, or separately, act as a tissue-selective partialagonist. For example, a multibinding compound with β2 adrenoceptoragonist activity may exhibit a full maximal response in relaxing smoothmuscle cells in bronchial tissue but exhibit a partial maximal responseto adrenoceptor agonism in cardiac tissue. Thus, a multibinding compoundthat is a tissue-selective partial agonist may provide a lower incidenceof undesired side effects such as positive chronotropism and increasesin cardiac output.

[0229] The term “potency” refers to the minimum concentration at which aligand is able to achieve a desirable biological or therapeutic effect.The potency of a ligand is typically proportional to its affinity forits ligand binding site. In some cases, the potency may be non-linearlycorrelated with its affinity. In comparing the potency of two drugs,e.g., a multibinding agent and the aggregate of its unlinked ligand, thedose-response curve of each is determined under identical testconditions (e.g., in an in vitro or in vivo assay, in an appropriateanimal model such a human patient). The finding that the multibindingagent produces an equivalent biological or therapeutic effect at a lowerconcentration than the aggregate unlinked ligand is indicative ofenhanced potency.

[0230] The term “selectivity” or “specificity” is a measure of thebinding preferences of a ligand for different ligand binding sites(receptors). The selectivity of a ligand with respect to its targetligand binding site relative to another ligand binding site is given bythe ratio of the respective values of K_(d) (i.e., the dissociationconstants for each ligand-receptor complex) or, in cases where abiological effect is observed below the K_(d), the ratio of therespective EC₅₀'s (i.e., the concentrations that produce 50% of themaximum response for the ligand interacting with the two distinct ligandbinding sites (receptors)).

[0231] The term “ligand binding site” denotes the site on theβ-adrenergic receptor that recognizes a ligand domain and provides abinding partner for the ligand. The ligand binding site may be definedby monomeric or multimeric structures. This interaction may be capableof producing a unique biological effect, for example, agonism,antagonism, and modulatory effects or it may maintain an ongoingbiological event, and the like.

[0232] It should be recognized that the ligand binding sites of thereceptor that participate in biological multivalent binding interactionsare constrained to varying degrees by their intra- and inter-molecularassociations. For example, ligand binding sites may be covalently joinedto a single structure, noncovalently associated in a multimericstructure, embedded in a membrane or polymeric matrix, and so on andtherefore have less translational and rotational freedom than if thesame structures were present as monomers in solution.

[0233] The terms “agonism”, “partial agonism”, and “antagonism” are wellknown in the art. The term “modulatory effect” refers to the ability ofthe ligand to change the activity of an agonist or antagonist throughbinding to a ligand binding site.

[0234] The term “inert organic solvent” or “inert solvent” means asolvent which is inert under the conditions of the reaction beingdescribed in conjunction therewith including, by way of example only,benzene, toluene, acetonitrile, tetrahydrofuran, dimethylformamide,chloroform, methylene chloride, diethyl ether, ethyl acetate, acetone,methylethyl ketone, methanol, ethanol, propanol, isopropanol, t-butanol,dioxane, pyridine, and the like. Unless specified to the contrary, thesolvents used in the reactions described herein are inert solvents.

[0235] The term “treatment” refers to any treatment of a pathologiccondition in a mammal, particularly a human, and includes:

[0236] (i) preventing the pathologic condition from occurring in asubject which may be predisposed to the condition but has not yet beendiagnosed with the condition and, accordingly, the treatment constitutesprophylactic treatment for the disease condition;

[0237] (ii) inhibiting the pathologic condition, i.e., arresting itsdevelopment;

[0238] (iii) relieving the pathologic condition, i.e., causingregression of the pathologic condition; or

[0239] (iv) relieving the conditions mediated by the pathologiccondition.

[0240] The term “pathologic condition which is modulated by treatmentwith a ligand” covers all disease states (i.e., pathologic conditions)which are generally acknowledged in the art to be usefully treated witha ligand for the β2-adrenergic receptor in general, and those diseasestates which have been found to be usefully treated by a specificmultibinding compound of our invention. Such disease states include, byway of example only, the treatment of a mammal afflicted with asthma,chronic bronchitis, chronic pulmonary obstructive disease, and the like.

[0241] The term “therapeutically effective amount” refers to that amountof multibinding compound which is sufficient to effect treatment, asdefined above, when administered to a mammal in need of such treatment.The therapeutically effective amount will vary depending upon thesubject and disease condition being treated, the weight and age of thesubject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art.

[0242] The term “linker”, identified where appropriate by the symbol‘X’, refers to a group or groups that covalently attaches from 2 to 10ligands (as identified above) in a manner that provides for a compoundcapable of multivalency. Among other features, the linker is aligand-orienting entity that permits attachment of at least two copiesof a ligand (which may be the same or different) thereto. Additionally,the linker can be either a chiral or achiral molecule. In some cases,the linker maybe a covalent bond that attaches the ligands in a mannerthat provides for a compound capable of multivalency. Additionally, insome cases, the linker may itself be biologically active. The term“linker” does not, however, extend to cover solid inert supports such asbeads, glass particles, fibers, and the like. But it is understood thatthe multibinding compounds of this invention can be attached to a solidsupport if desired. For example, such attachment to solid supports canbe made for use in separation and purification processes and similarapplications.

[0243] The extent to which multivalent binding is realized depends uponthe efficiency with which the linker or linkers that joins the ligandspresents these ligands to the array of available ligand binding sites.Beyond presenting these ligands for multivalent interactions with ligandbinding sites, the linker or linkers spatially constrains theseinteractions to occur within dimensions defined by the linker orlinkers. Thus, the structural features of the linker (valency, geometry,orientation, size, flexibility, chemical composition, etc.) are featuresof multibinding agents that play an important role in determining theiractivities.

[0244] The linkers used in this invention are selected to allowmultivalent binding of ligands to the ligand binding sites of a β2adrenergic receptor, whether such sites are located interiorly, bothinteriorly and on the periphery of the receptor structure, or at anyintermediate position thereof.

[0245] Representative Compounds of Formula (I)

[0246] I. Representative multibinding compounds of Formula (I) wherein pis 2, q is 1, Ar¹ is 4-hydroxy-3-hydroxymethylphenyl, Ar² is1,4-phenylene, R¹ and R² are hydrogen, X, W, Q. and Ar³ are as definedin Table A below are: TABLE A Stereo- Cpd. chem. at # *C W X -Q-Ar³ (**= stereochem)  1A (RS) —(CH₂)₂— bond —NH—CH₂—** CH(OH)phenyl ** = (S) 2A (RS) —(CH₂)₂— bond —NH—CH₂—** CH(OH)phenyl ** = (R)  3A (RS)—(CH₂)₂— bond —NH—CH₂—** CH(OH)phenyl ** = (RS)  4A (AS) —(CH₂)₂— bond—NH—CH₂—** CH(OH)-(4-hydroxy-3- hydroxy-methyl)phenyl ** = (RS)  5A (RS)—(CH₂)₆O— bond —(CH₂)₃—O—(CH₂)₆—NH—CH₂—** CH(OH)-(4-hydroxy-3-hydroxyethyl)phenyl ** = (RS)  6A (RS) —CH₂— bond —NH—CH₂—**CH(OH)-(4-hydroxy-3- hydroxy-methyl)phenyl ** = (RS)  7A (R) —(CH₂)₂—bond —NH—CH₂—** CH(OH)phenyl ** = (S)  8A (R) —(CH₂)₂— bond —NH—CH₂—**CH(OH)phenyl ** = (R)  9A (RS) —(CH₂)₆—O— bond—O—(CH₂)₆—O-[4-(3-hydroxypropyly]- (CH₂)₃ phenyl 10A (RS) —CH₂*CH(OH)—bond —O—(CH₂)—** CH(OH)—(CH₂)—NH—CH₂— CH₂—O— * = (RS) **CH(OH)-(4-hydroxy-3-hydroxy- methyl)phenyl ** = (RS) 11A (RS) —(CH₂)₂—bond —NH—CH₂—** CH(OH)—O-naphth-1-yl ** = (RS)

[0247] II. Representative multibinding compounds of Formula (I) whereinp is 2, q is 1, Ar¹ is 4-hydroxy-3-hydroxymethylphenyl, Ar² is1,4-phenylene, R¹ and R² are hydrogen, X, W, Q, and Ar^(3,) are asdefined Table B below are: TABLE B Stereo- Cpd. chem. at # *C W X Q —Ar³1B (RS) bond —O-(p-C₆H₄)—NH—CH₂— bond 4-hydroxy-3- ** CH(OH)— ** = (RS)hydroxymethyl phenyl 2B (RS) bond —O— bond 4-aminophenyl 3B (RS)—(CH₂)₆— —O—(CH₂)₁₀—O-(p-C₆H₄)— bond 4-hydroxy-3- O— (CH₂)₃—O—(CH₂)₆—NH—hydroxy- (CH₂)₃— CH₂—** CH(OH)— methylphenyl ** = (RS) stereochem. 4B(RS) —(CH₂)₆— —O—(CH₂)₆—O-(p-C₆H₄)— bond 4-hydroxy-3- O—(CH₂)₃—O—(CH₂)₅—NH— hydroxy- (CH₂)₃— CH₂—** CH(OH)— methylphenyl ** =(RS) stereochem. 5B (RS) —(CH₂)₂— —O—(CH₂)₄— bond phenyl

[0248] III. Representative multibinding compounds of Formula (I) whereinp is 2, q is 1, Ar¹ is 4-hydroxy-3-hydroxy-methylphenyl, R¹ and R² arehydrogen, Ar³ is (4-hydroxy-3-hydroxymethyl)phenyl, X, W, Q, and Ar² areas defined in Table C below are: TABLE C Cpd. Stereochem. # at *C W XAr² Q 1C (RS) bond bond trans-1,4- —NH—CH₂—** CH(OH)— cyclohexane ** =(RS) 2C (RS) —CH₂— bond 1,3- —CH₂—NH—CH₂—** cyclohexane CH(OH)— ** =(RS) 3C (RS) —(CH₂)₃— bond 1,4-piperazine —(CH₂)₃—NH—CH₂—** CH(OH)— ** =(RS) 4C (RS) bond bond p-menthane —NH—CH₂— ** CH(OH)— ** = (RS) 5C (RS)bond bond 1,2-phenylene —CH₂—NH—CH₂—** CH(OH)— ** = (RS)

[0249] IV. Representative multibinding compounds of Formula (I) whereinp is 2, q is 1, Ar¹ and Ar³ are 4-hydroxy-3-hydroxymethylphenyl, R¹ andR² are hydrogen, Q is a bond, and W, Ar², and X are as defined in TableD below are: TABLE D Cpd. Stereochem. # at *C W Ar² X 1D (RS) bond 1,4-—(CH₂)-(p-C₆H₁₀)—NH—CH₂— cyclohexane ** CH(OH)— ** = (RS) stereochem.

[0250] V. Representative multibinding compounds of Formula (I) wherein pis 2, q is 1, R¹ and R² are hydrogen, W is —(CH₂)₂—, Ar² is1,4-phenylene, —Q—Ar³, is [2-hydroxy-2-phenyl]ethylamino, X is a bondand Ar¹ is as defined below are as shown in Table E below: TABLE EStereochem. Stereochem. Cpd. # Ar¹ at *C at **C 1E phenyl (RS) (RS) 2Ephenyl (R) (S) 3E phenyl (R) (R) 4E 4-amino-3,5-dichlorophenyl (RS) (RS)5E 4-amino-3,5-dichlorophenyl (R) (R) 6E 4-amino-3,5-dichlorophenyl (S)(S) 7E 4-amino-3,5-dichlorophenyl (R) (S) 8E 4-amino-3,5-dichlorophenyl(S) (R) 9E 3-formyl-amino-4-hydroxyphenyl (RS) (RS) 10E 3-formyl-amino-4-hydroxyphenyl (R) (R) 11E 3-formyl-amino-4-hydroxyphenyl (S) (S) 12E 3-formyl-amino-4-hydroxyphenyl (R) (S) 13E 3-formyl-amino-4-hydroxyphenyl (S) (R)

[0251] VI. Miscellanous compounds:

PREFERRED EMBODIMENTS

[0252] While the broadest definition of this invention is set forth inthe Summary of the Invention, certain compounds of Formula (I) arepreferred.

[0253] (A) A preferred group is a multibinding compound of Formula (II):

[0254] where *C has RS, R, or S stereochemistry; Within this group (A) amore preferred group of compounds is that wherein:

[0255] (i) Ar¹ is aryl, more preferably Ar¹ is a phenyl ring of formula(c):

[0256] wherein:

[0257] R⁴ is hydrogen, alkyl, halo, or alkoxy, preferably hydrogen,methyl, fluoro, chloro, or methoxy;

[0258] R⁵ is hydrogen, hydroxy, halo, halo, amino, or —NHSO₂R^(a) whereR^(a) is alkyl, preferably hydrogen, hydroxy, fluoro, chloro, amino, or—NHSO₂CH₃; and

[0259] R⁶ is hydrogen, halo, hydroxy, alkoxy, substituted alkyl,sulfonylamino, aminoacyl, or acylamino; preferably hydrogen, chloro,fluoro, hydroxy, methoxy, hydroxymethyl, —CH₂SO₂CH₃, —NHSO₂CH₃, —NHCHO,or —CONH₂, or —NHCONH₂;

[0260] (ii) Ar¹ is heteroaryl, more preferably Ar¹ is2,8-dihydroxyquinolin-5-yl or 3-bromoisoxazol-5-yl; or

[0261] (iii) Ar¹ is heterocyclyl, more preferably Ar¹ is heterocyclylfused to an aryl ring, most preferably 6-fluorochroman-2-yl;

[0262] W is a bond linking the —NR²— group to Ar², alkylene, or asubstituted alkylene group wherein one or more of the carbon atoms inthe alkylene and the substituted alkylene group is optionally replacedby —O—, preferably a covalent bond, methylene, ethylene, propylene,—(CH₂)₆—O—(CH₂)₃—, —(CH₂)₆—O—, or —CH₂CH(OH)CH₂—O—; and

[0263] Ar² is phenyl wherein the W and the X groups are attached at the1,2-, 1,3-, and 1,4-positions of the phenyl ring; cyclohexyl optionallysubstituted with methyl and wherein the W and the X groups are attachedat the 1,3- and 1,4-positions of the cyclohexyl ring; or piperazinewherein the W and the X groups are attached at the 1,4-positions of thepiperazine ring, preferably 1,4-phenylene.

[0264] Within the above more preferred groups, even more preferredgroups of compounds are wherein:

[0265] (a) X is —O—, —O-alkylene, —O-(arylene)-NH-(substitutedalkylene)-,—O-(alkylene)-O-(arylene)-(alkylene)-O-(alkylene)-NH-(substitutedalkylene)-, —O-(alkylene)-O-(arylene)—, or-(alkylene)-(cycloalkylene)-NH-(substituted alkylene)-, preferably—O—(CH₂)₄—; —CH₂-(1,4-cyclohexyl)-NH—CH₂—CH(OH)—;—O-(1,4-phenylene)-NH—CH₂—CH(OH)—;—O—(CH₂)₁₀—O-(1,4-phenylene)-(CH₂)₃—O—(CH₂)₆—NH—CH₂—CH(OH)—;—O—(CH₂)₆—O-(1,4-phenylene)-(CH₂)₃—O—(CH₂)₅—NH—CH₂—CH(OH)—; or—O—(CH₂)₆—O-(1,4-phenylene)-; and

[0266] Q is a covalent bond; or

[0267] (b) X is a bond; and

[0268] Q is a substituted alkylene group wherein one or more of thecarbon atoms in said substituted alkylene group is optionally replacedby a heteroatom selected from the group consisting of —NR^(a)— (whereR^(a) is hydrogen, alkyl, or acyl) and —O—, preferably—NH—CH₂—**CH(OH)—; —NH—CH₂—**CH(OH)—CH₂—O—; —NH—**CH(CH₂OH)—;—CH₂—NH—CH₂—**CH(OH)—; —C(CH₃)₂—NH—CH₂—**CH(OH)—;—(CH₂)₃—NH—CH₂—**CH(OH)—; —(CH₂)₃—O—(CH₂)₆—NH—CH₂—**CH(OH)—;—(CH₂)₂—NH—CH₂—**CH(OH)—; —O—CH₂—**CH(OH)—; or —NH—CH₂—**CH(OH)—CH₂—O—;more preferably —NH—CH₂—**CH(OH)—; —NH—**CH(CH₂OH)—;—(CH₂)₃—O—(CH₂)₆—NH—CH₂—**CH(OH)—; or —NH—CH₂—**CH(OH)—CH₂—O— (where **is RS, R or S stereochemistry), most preferably —NH—**CH(CH₂OH)— where** is RS, R or S stereochemistry;

[0269] Within the above preferred, more preferred group of compounds, aparticularly preferred group of compounds is that wherein:

[0270] (i) Ar³ is same as Ar¹ as defined in preferred embodiments(A)(i)-(iii) above.

[0271] Another particularly preferred group of compounds is thatwherein:

[0272] (ii) Ar³ is a phenyl ring of formula (d):

[0273] wherein:

[0274] R⁷ is hydrogen, alkyl, alkenyl, substituted alkyl, halo, alkoxy,substituted alkoxy, hydroxy, aminoacyl, or heteroaryl, preferablyhydrogen, methyl, propen-2-yl, fluoro, chloro, methoxy, —OCH₂CO₂Me,—OCON(CH₃)₂, hydroxy, —CH₂CONH₂, —NHCOCH₃, —NHCHO, imidazol-1-yl, or1-methyl-4-trifluoromethylimidazol-2-yl; and

[0275] R⁸ is hydrogen, halo, alkoxy, substituted alkoxy, or acylamino,preferably hydrogen, fluoro, chloro, methoxy, —OCH₂CO₂Me, —OCON(CH₃)₂,—NHCHO, or —CONH₂.

[0276] (iii) Yet another particularly preferred group of compounds isthat wherein:

[0277] Ar₃ is naphthyl, pyridyl, benzimidazol-1-yl, indolyl,2-cyanoindolyl, carbazolyl, 4-methylindanyl,5-(CH₃CO₂CH₂O—)-1,2,3,4-tetrahydronaphthyl, 1H-2-oxoindole,2,3,4-trihydrothianaphthalene, 4-hydroxy-2-benzothiazolinone, or4-oxo-2,3-dihydrothianapthalene.

[0278] Within the above preferred, more preferred, and particularlypreferred groups, even more particularly preferred group is thatwherein:

[0279] Ar¹ is phenyl, 4-hydroxyphenyl, 3,4-dihydroxyphenyl,3,4-dichlorophenyl, 3,5-dihydroxyphenyl, 2-chloro-3,4-dihydroxyphenyl,2-fluoro-3,4-dihydroxyphenyl, 2-chloro-3,5-dihydroxyphenyl,2-fluoro-3,5-dihydroxyphenyl, 4-hydroxy-3-methoxyphenyl,4-hydroxy-3-hydroxymethylphenyl, 4-hydroxy-3-(HCONH—)phenyl,4-hydroxy-3-(NH₂CO—)phenyl, 3-chlorophenyl, 2,5-dimethoxyphenyl,4-(CH₃SO₂NH—)-phenyl, 4-hydroxy-3-(CH₃SO₂CH₂—)phenyl,4-hydroxy-3-(CH₃SO₂NH—)phenyl, 4-hydroxy-3-(NH₂CONH—)phenyl,3,5-dichloro-4-aminophenyl,

[0280] preferably 4-hydroxy-3-hydroxymethylphenyl,4-hydroxy-3-(HCONH—)phenyl, 3,5-dichloro-4-aminophenyl, or

[0281] most preferably 4-hydroxy-3-(HCONH—)phenyl or3,5-dichloro-4-aminophenyl; and

[0282] Ar³ is:

[0283] preferably, phenyl or 4-hydroxy-3-hydroxymethylphenyl, morepreferably phenyl.

GENERAL SYNTHETIC SCHEME

[0284] Compounds of this invention can be made by the methods depictedin the reaction schemes shown below.

[0285] The starting materials and reagents used in preparing thesecompounds are either available from commercial suppliers such as AldrichChemical Co., (Milwaukee, Wis., USA), Bachem (Torrance, Calif., USA),Emka-Chemie, or Sigma (St. Louis, Mo., USA) or are prepared by methodsknown to those skilled in the art following procedures set forth inreferences such as Fieser and Fieser's Reagents for Organic Synthesis,Volumes 1-15 (John Wiley and Sons, 1991); Rodd's Chemistry of CarbonCompounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers,1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991),March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition),and Larock's Comprehensive Organic Transformations (VCH Publishers Inc.,1989).

[0286] The starting materials and the intermediates of the reaction maybe isolated and purified if desired using conventional techniques,including but not limited to filtration, distillation, crystallization,chromatography, and the like. Such materias may be characterized usingconventional means, including physical constants and spectral data.

[0287] Furthermore, it will be appreciated that where typical orpreferred process conditions (i.e., reaction temperatures, times, moleratios of reactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

[0288] Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group as well assuitable conditions for protection and deprotection are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Second Edition, Wiley, N.Y., 1991, andreferences cited therein.

[0289] These schemes are merely illustrative of some methods by whichthe compounds of this invention can be synthesized, and variousmodifications to these schemes can be made and will be suggested to oneskilled in the art having referred to this disclosure.

Preparation of a Multibinding Compound of Formula (I)

[0290] In general, a multibinding compound of Formula (I) where p is 2and q is 1 can be prepared as illustrated and described in Schemes A-Dbelow.

[0291] A multibinding compound of Formula (I) where p is 2 and q is 1can be prepared by covalently attaching the ligands, L, wherein at leastone of the ligand is selected from a compound of formula (a) as definedin the Summary of the Invention, to a linker, X, as shown in Scheme Abelow.

[0292] In method (a), a multibinding compound of Formula (I) where p is2 and q is 1 is prepared in one step, by covalently attaching theligands, L, to a linker, X, where FG¹ and FG² represent a functionalgroup such as halo, amino, hydroxy, thio, aldehyde, ketone, carboxy,carboxy derivatives such as acid halide, ester, amido, and the like.This method is preferred for preparing compounds of Formula (I) wherethe ligands are the same.

[0293] In method (b), the compounds of Formula (I) are prepared in astepwise manner by covalently attaching one equivalent of a ligand, L₁,with a ligand X where where FG¹ and FG² represent a functional group asdefined above, and FG²PG is a protected functional group to give anintermediate of formula (II). Deprotection of the second functionalgroup on the ligand, followed by reaction with a ligand L₂, which may besame or different than ligand L₁, then provides a compound of Formula(I). This method is suitable for preparing compounds of Formula (I)where the ligands are the non-identical.

[0294] The ligands are covalently attached to the linker usingconventional chemical techniques providing for covalent linkage of theligand to the linker. Reaction chemistries resulting in such linkagesare well known in the art and involve the use of complementaryfunctional groups on the linker and ligand as shown in Table I below.TABLE I Representative Complementary Binding Chemistries First ReactiveGroup Second Reactive Group Linkage carboxyl amine amide sulfonyl halideamine sulfonamide hydroxyl alkyl/aryl halide ether hydroxyl isocyanateurethane amine epoxide β-hydroxyamine amine alkyl/aryl halide alkylamineamine isocyanate urea hydroxyl carboxyl ester amine aldehyde amine

[0295] Reaction between a carboxylic acid of either the linker or theligand and a primary or secondary amine of the ligand or the linker inthe presence of suitable, well-known activating agents such asdicyclohexylcarbodiimide, results in formation of an amide bondcovalently linking the ligand to the linker; reaction between an aminegroup of either the linker or the ligand and a sulfonyl halide of theligand or the linker, in the presence of a base such as triethylamine,pyridine, an the like results in formation of a sulfonamide bondcovalently linking the ligand to the linker; and reaction between analcohol or phenol group of either the linker or the ligand and an alkylor aryl halide of the ligand or the linker in the presence of a basesuch as triethylamine, pyridine, and the like, results in formation ofan ether bond covalently linking the ligand to the linker.

[0296] A multibinding compound of Formula (I) where p is 2 q is 1, thesecond ligand A³ is the same as Ar¹, X is a bond, and Q is2-hydroxyethylamino group, and the ligands are linked through the Ar²group can be prepared from an aryl glyoxal derivative of formula 1 asshown in Scheme B below.

[0297] Condensation of an acetophenone derivative of formula 1 with adiamine of formula 2 in an ethereal solution such as tetrahydrofuranprovides an imine of formula 3. Reduction of the imine with a suitablereducing agent such as diborane provides a compound of Formula (I).Suitable reaction solvents are tetrahydrofuran, and the like. Compound 1where Ar¹ is phenyl is prepared by heating acetophenone in 48%hydrobromic acid in dinethylsulfoxide.

[0298] Compounds of formula 1 can be prepared by methods well known inthe art. For example,α,α-dihydroxy-4-hydroxy-3-methoxycarbonylacetophenone can be prepared byheating 5-acetylsalicylic acid methyl ester in 48% hydrobromic acid.

[0299] Alternatively, a multibinding compound of Formula (I) where p is2, q is 1, the second ligand Ar³ is the same as Ar¹, X is a bond, and Qis 2-hydroxyethylamino group, and the ligands are linked through the Ar²group can be prepared from an aryl epoxide of formula 4 as shown inScheme C below.

[0300] A compound of Formula (I) can be prepared by reacting an epoxideof formula 4 with a diamine of formula 2. Epoxides 4 are eithercommercially available or they can be prepared by the methods describedin Kierstead, R. W. et. al. J. Med. Chem. 26, 1561-1569, (1983) or Hett,R. et. al. Tet. Lett. 35, 9345-9348 (1994).

[0301] Another method of preparing a multibinding compound of Formula(I) where p is 2, q is 1, the second ligand Ar³ is the same as Ar¹, X isa bond, and Q is 2-hydroxyethylamino group, and the ligands are linkedthrough the Ar² group can be prepared from an acetophenone derivative offormula 5 as shown in Scheme D below.

[0302] Bromination of an acetophenone derivative of formula 5 withbromine in a halogenated organic solvent such as chloroform provides anα-bromoacetophenone derivative of formula 6. Treatment of 6 with sodiumazide followed by reduction of the resulting azide 7 with a suitablereducing agent such as lithium aluminum hydride provides ethanolaminederivative of formula 8. Condensation of two equivalents of 8 with adialdehyde compound of formula 9 provides an imine of formula 10 whichis converted to a compound of Formula (I) as described in Scheme Aabove.

[0303] Another method of preparing a multibinding compound of Formula(I) where p is 2, q is 1, Ar¹ and Ar³ are different, X is a bond, and Qis 2-hydroxyethylamino group, and the ligands are linked through the Ar²group can be prepared as shown in Scheme E below.

[0304] Condensation of a diamine of formula 11 (where PG₁ and PG₂ aresuitable amino protecting groups which can be selectively removed) witha glyoxal of formula 2 followed by reduction of the resulting imine offormula 12 with a suitable reducing agent such as diborane in a suitablereaction solvents such as tetrahydrofuran provides a compound of formula13. Compounds of formula 11 can be prepared by methods described in FIG.14.

[0305] Reaction of compound 15 with an alpha bromoacetophenone compoundof formula 6 followed by reduction of the keto group provides a compoundof formula 16. The reaction is carried out under conditions well knownin the art. Deprotection of the amino protecting group then provides acompound of Formula (I). The deprotection reaction conditions depend onthe nature of the protecting group. For example, if the protecting groupis benzyl, it is removed under catalytic hydrogenation reactionconditions.

[0306] Any compound which is a β2 adrenergic receptor agonist can beused as a ligand in this invention. Typically, a compound selected foruse as a ligand will have at least one functional group, such as anamino, hydroxyl, thiol or carboxyl group and the like, which allows thecompound to be readily coupled to the linker. Compounds having suchfunctionality are either known in the art or can be prepared by routinemodification of known compounds using conventional reagents andprocedures.

[0307] Linkers can be attached to different positions on the ligandmolecule to achieve different orientations of the ligand domains, andthereby facilitate multivalency. While a number of positions onβ-adrenergic-modulating ligands are synthetically practical for linking,it is preferred to preserve those ligand substructures which are mostimportant for ligand-receptor binding. At present, the aryl group andthe sidechain nitrogen are preferred points of attachment.

[0308] It will be apparent to one skilled in the art that the abovechemistries are not limited to preparing bivalent multibinding compoundsof Formula (I) and can be used to prepare tri-, tetra-, etc.,multibinding compounds of Formula (I).

[0309] The linker is attached to the ligand at a position that retainsligand domain-ligand binding site interaction and specifically whichpermits the ligand domain of the ligand to orient itself to bind to theligand binding site. Such positions and synthetic protocols for linkageare well known in the art. The term linker embraces everything that isnot considered to be part of the ligand.

[0310] The relative orientation in which the ligand domains aredisplayed derives from the particular point or points of attachment ofthe ligands to the linker, and on the framework geometry. Thedetermination of where acceptable substitutions can be made on a ligandis typically based on prior knowledge of structure-activityrelationships (SAR) of the ligand and/or congeners and/or structuralinformation about ligand-receptor complexes (e.g., X-raycrystallography, NMR, and the like). Such positions and the syntheticmethods for covalent attachment are well known in the art. Followingattachment to the selected linker (or attachment to a significantportion of the linker, for example 2-10 atoms of the linker), theunivalent linker-ligand conjugate may be tested for retention ofactivity in the relevant assay.

[0311] The linker, when covalently attached to multiple copies of theligands, provides a biocompatible, substantially non-immunogenicmultibinding compound. The biological activity of the multibindingcompound is highly sensitive to the valency, geometry, composition,size, flexibility or rigidity, etc. of the linker and, in turn, on theoverall structure of the multibinding compound, as well as the presenceor absence of anionic or cationic charge, the relativehydrophobicity/hydrophilicity of the linker, and the like on the linker.Accordingly, the linker is preferably chosen to maximize the biologicalactivity of the multibinding compound. The linker may be chosen toenhance the biological activity of the molecule. In general, the linkermay be chosen from any organic molecule construct that orients two ormore ligands to their ligand binding sites to permit multivalency. Inthis regard, the linker can be considered as a “framework” on which theligands are arranged in order to bring about the desiredligand-orienting result, and thus produce a multibinding compound.

[0312] For example, different orientations can be achieved by includingin the framework groups containing mono- or polycyclic groups, includingaryl and/or heteroaryl groups, or structures incorporating one or morecarbon-carbon multiple bonds (alkenyl, alkenylene, alkynyl or alkynylenegroups). Other groups can also include oligomers and polymers which arebranched- or straight-chain species. In preferred embodiments, rigidityis imparted by the presence of cyclic groups (e.g., aryl, heteroaryl,cycloalkyl, heterocyclic, etc.). In other preferred embodiments, thering is a six or ten member ring. In still further preferredembodiments, the ring is an aromatic ring such as, for example, phenylor naphthyl.

[0313] Different hydrophobic/hydrophilic characteristics of the linkeras well as the presence or absence of charged moieties can readily becontrolled by the skilled artisan. For example, the hydrophobic natureof a linker derived from hexamethylene diamine (H₂N(CH₂)₆NH₂) or relatedpolyamines can be modified to be substantially more hydrophilic byreplacing the alkylene group with a poly(oxyalkylene) group such asfound in the commercially available “Jeffamines”.

[0314] Different frameworks can be designed to provide preferredorientations of the ligands. Such frameworks may be represented by usingan array of dots (as shown below) wherein each dot may potentially be anatom, such as C, O, N, S, P, H, F, Cl, Br, and F or the dot mayalternatively indicate the absence of an atom at that position. Tofacilitate the understanding of the framework structure, the frameworkis illustrated as a two dimensional array in the following diagram,although clearly the framework is a three dimensional array in practice:

[0315] Each dot is either an atom, chosen from carbon, hydrogen, oxygen,nitrogen, sulfur, phosphorus, or halogen, or the dot represents a pointin space (i.e., an absence of an atom). As is apparent to the skilledartisan, only certain atoms on the grid have the ability to act as anattachment point for the ligands, namely, C, O, N, S and P.

[0316] Atoms can be connected to each other via bonds (single, double ortriple bonds with acceptable resonance and tautomeric forms), withregard to the usual constraints of chemical bonding. Ligands may beattached to the framework via single, double or triple bonds (withchemically acceptable tautomeric and resonance forms). Multiple ligandgroups (2 to 10) can be attached to the framework such that the minimal,shortest path distance between adjacent ligand groups does not exceed100 atoms. Preferably, the linker connections to the ligand is selectedsuch that the maximum spatial distance between two adjacent ligands isno more than 100 Å.

[0317] An example of a linker as presented by the grid is shown belowfor a biphenyl construct.

[0318] Nodes (1, 2), (2, 0), (4, 4), (5, 2), (4, 0), (6, 2), (7, 4), (9,4), (10, 2), (9, 0), (7, 0) all represent carbon atoms. Node (10, 0)represents a chlorine atom. All other nodes (or dots) are points inspace (i.e., represent an absence of atoms).

[0319] Nodes (1, 2) and (9, 4) are attachment points. Hydrogen atoms areaffixed to nodes (2, 4), (4, 4), (4, 0), (2, 0), (7, 4), (10, 2) and (7,0). Nodes (5, 2) and (6, 2) are connected by a single bond.

[0320] The carbon atoms present are connected by either a single ordouble bonds, taking into consideration the principle of resonanceand/or tautomerism.

[0321] The intersection of the framework (linker) and the ligand group,and indeed, the framework (linker) itself can have many differentbonding patterns. Examples of acceptable patterns of three contiguousatom arrangements are shown in the following diagram: CCC NCC OCC SCCPCC CCN NCN OCN SCN PCN CCO NCO OCO SCO PCO CCS NCS OCS SCS PCS CCP NCPOCP SCP PCP CNC NNC ONC SNC PNC CNN NNN ONN SNN PNN CNO NNO ONO SNO PNOCNS NNS ONS SNS PNS CNP NNP ONP SNP PNP COC NOC OOC SOC POC COO NON OONSON PON COC NOO OOO SOO POO COP NOP OOS SOS POS CSC NSC OOP SOP POP CSNNSN OSC SSC PSC CSO NSO OSN SSN PSN CSS NSS 0SO SSO PSO CSP NSP OSS SSSPSS CPC NPC OSP SSP PSP CPN NPN OPC SPC PPC CPO NPO OPN SPN PPN CPS NPSOPO SPO PPO CPP NPP OPS SPS PPS OPP SPP PPP

[0322] One skilled in the art would be able to identify bonding patternsthat would produce multivalent compounds. Methods for producing thesebonding arrangements are described in March, “Advanced OrganicChemistry”, 4th Edition, Wiley-Tnterscience, New York, N.Y. (1992).These arrangements are described in the grid of dots shown in the schemeabove. All of the possible arrangements for the five most preferredatoms are shown. Each atom has a variety of acceptable oxidation states.The bonding arrangements underlined are less acceptable and are notpreferred.

[0323] Examples of molecular structures in which the above bondingpatterns could be employed as components of the linker are shown below.

[0324] identification of a appropriated frameworks geometry and size ofligand domain presentation are important steps in the construction of amultibinging compound with enhanced activity. Systematic spatialsearching strategies can be used to aid in the identification ofpreferred frameworks through an iterative process. FIG. 3 illustrates auseful strategy for determining an optimal framework display orientationfor ligand domains. Various other strategies are known to those skilledin the art of molecular design and can be used for preparing compoundsof this invention.

[0325] As shown in FIG. 1, display vectors around similar central corestructures such as a phenyl structure (Panel A) and a cyclohexanestructure (Panel B) can be varied, as can the spacing of the liganddomain from the core structure (i.e., the length of the attachingmoiety). It is to be noted that core structures other than those shownhere can be used for determining the optimal framework displayorientation of the ligands. The process may require the use of multiplecopies of the same central core structure or combinations of differenttypes of display cores.

[0326] The above-described process can be extended to trimers (FIG. 2)and compound of higher valency (FIGS. 3 and 4).

[0327] Assays of each of the individual compounds of a collectiongenerated as described above will lead to a subset of compounds with thedesired enhanced activities (e.g., potency, selectivity, etc.). Theanalysis of this subset using a technique such as Ensemble MolecularDynamics will provide a framework orientation that favors the propertiesdesired. A wide diversity of linkers is commercially available (see,e.g., Available Chemical Directory (ACD)). Many of the linkers that aresuitable for use in this invention fall into this category. Other can bereadily synthesized by methods well known in the art and/or aredescribed below.

[0328] Having selected a preferred framework geometry, the physicalproperties of the linker can be optimized by varying the chemicalcomposition thereof The composition of the linker can be varied innumerous ways to achieve the desired physical properties for themultibinding compound.

[0329] It can therefore be seen that there is a plethora ofpossibilities for the composition of a linker. Examples of linkersinclude aliphatic moieties, aromatic moieties, steroidal moieties,peptides, and the like. Specific examples are peptides or polyamides,hydrocarbons, aromatic groups, ethers, lipids, cationic or anionicgroups, or a combination thereof.

[0330] Examples are given below, but it should be understood thatvarious changes may be made and equivalents may be substituted withoutdeparting from the true spirit and scope of the invention. For example,properties of the linker can be modified by the addition or insertion ofancillary groups into or onto the linker, for example, to change thesolubility of the multibinding compound (in water, fats, lipids,biological fluids, etc.), hydrophobicity, hydrophilicity, linkerflexibility, antigenicity, stability, and the like. For example, theintroduction of one or more poly(ethylene glycol) (PEG) groups onto orinto the linker enhances the hydrophilicity and water solubility of themultibinding compound, increases both molecular weight and molecularsize and, depending on the nature of the unPEGylated linker, mayincrease the in vivo retention time. Further PEG may decreaseantigenicity and potentially enhances the overall rigidity of thelinker.

[0331] Ancillary groups which enhance the watersolubility/hydrophilicity of the linker and, accordingly, the resultingmultibinding compounds are useful in practicing this invention. Thus, itis within the scope of the present invention to use ancillary groupssuch as, for example, small repeating units of ethylene glycols,alcohols, polyols (e.g., glycerin, glycerol propoxylate, saccharides,including mono-, oligosaccharides, etc.), carboxylates (e.g., smallrepeating units of glutamic acid, acrylic acid, etc.), amines (e.g.,tetraethylenepentamine), and the like) to enhance the water solubilityand/or hydrophilicity of the multibinding compounds of this invention.In preferred embodiments, the ancillary group used to improve watersolubility/hydrophilicity will be a polyether .

[0332] The incorporation of lipophilic ancillary groups within thestructure of the linker to enhance the lipophilicity and/orhydrophobicity of the multibinding compounds described herein is alsowithin the scope of this invention. Lipophilic groups useful with thelinkers of this invention include, by way of example only, aryl andheteroaryl groups which, as above, may be either unsubstituted orsubstituted with other groups, but are at least substituted with a groupwhich allows their covalent attachment to the linker. Other lipophilicgroups useful with the linkers of this invention include fatty acidderivatives which do not form bilayers in aqueous medium until higherconcentrations are reached.

[0333] Also within the scope of this invention is the use of ancillarygroups which result in the multibinding compound being incorporated oranchored into a vesicle or other membranous structure such as a liposomeor a micelle. The term “lipid” refers to any fatty acid derivative thatis capable of forming a bilayer or a micelle such that a hydrophobicportion of the lipid material orients toward the bilayer while ahydrophilic portion orients toward the aqueous phase. Hydrophiliccharacteristics derive from the presence of phosphato, carboxylic,sulfato, amino, sulfhydryl, nitro and other like groups well known inthe art. Hydrophobicity could be conferred by the inclusion of groupsthat include, but are not limited to, long chain saturated andunsaturated aliphatic hydrocarbon groups of up to 20 carbon atoms andsuch groups substituted by one or more aryl, heteroaryl, cycloalkyl,and/or heterocyclic group(s). Preferred lipids are phosphglycerides andsphingolipids, representative examples of which includephosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidic acid, palmitoyleoylphosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoyl-phosphatidylcholine ordilinoleoylphosphatidylcholine could be used. Other compounds lackingphosphorus, such as sphingolipid and glycosphingolipid families are alsowithin the group designated as lipid. Additionally, the amphipathiclipids described above may be mixed with other lipids includingtriglycerides and sterols.

[0334] The flexibility of the linker can be manipulated by the inclusionof ancillary groups which are bulky and/or rigid. The presence of bulkyor rigid groups can hinder free rotation about bonds in the linker orbonds between the linker and the ancillary group(s) or bonds between thelinker and the functional groups. Rigid groups can include, for example,those groups whose conformational lability is restrained by the presenceof rings and/or multiple bonds within the group, for example, aryl,heteroaryl, cycloalkyl, cycloalkenyl, and heterocyclic groups. Othergroups which can impart rigidity include polypeptide groups such asoligo- or polyproline chains.

[0335] Rigidity can also be imparted electrostatically. Thus, if theancillary groups are either positively or negatively charged, thesimilarly charged ancillary groups will force the presenter linker intoa configuration affording the maximum distance between each of the likecharges. The energetic cost of bringing the like-charged groups closerto each other will tend to hold the linker in a configuration thatmaintains the separation between the like-charged ancillary groups.Further ancillary groups bearing opposite charges will tend to beattracted to their oppositely charged counterparts and potentially mayenter into both inter- and intramolecular ionic bonds. This non-covalentmechanism will tend to hold the linker into a conformation which allowsbonding between the oppositely charged groups. The addition of ancillarygroups which are charged, or alternatively, bear a latent charge whendeprotected, following addition to the linker, include deprotonation ofa carboxyl, hydroxyl, thiol or amino group by a change in pH, oxidation,reduction or other mechanisms known to those skilled in the art whichresult in removal of the protecting group, is within the scope of thisinvention.

[0336] Rigidity may also be imparted by internal hydrogen bonding or byhydrophobic collapse.

[0337] Bulky groups can include, for example, large atoms, ions (e.g.,iodine, sulfur, metal ions, etc.) or groups containing large atoms,polycyclic groups, including aromatic groups, non-aromatic groups andstructures incorporating one or more carbon-carbon multiple bonds (i.e.,alkenes and alkynes). Bulky groups can also include oligomers andpolymers which are branched- or straight-chain species. Species that arebranched are expected to increase the rigidity of the structure more perunit molecular weight gain than are straight-chain species.

[0338] In preferred embodiments, rigidity is imparted by the presence ofcyclic groups (e.g., aryl, heteroaryl, cycloalkyl, heterocyclic, etc.).In other preferred embodiments, the linker comprises one or moresix-membered rings. In still further preferred embodiments, the ring isan aryl group such as, for example, phenyl or naphthyl.

[0339] In view of the above, it is apparent that the appropriateselection of a linker group providing suitable orientation,restricted/unrestricted rotation, the desired degree ofhydrophobicity/hydrophilicity, etc. is well within the skill of the art.Eliminating or reducing antigenicity of the multibinding compoundsdescribed herein is also within the scope of this invention. In certaincases, the antigenicity of a multibinding compound may be eliminated orreduced by use of groups such as, for example, poly(ethylene glycol).

[0340] As explained above, the multibinding compounds described hereincomprise 2-10 ligands attached to a linker that attaches the ligands insuch a manner that they are presented to the enzyme for multivalentinteractions with ligand binding sites thereon/therein. The linkerspatially constrains these interactions to occur within dimensionsdefined by the linker. This and other factors increases the biologicalactivity of the multibinding compound as compared to the same number ofligands made available in monobinding form.

[0341] The compounds of this invention are preferably represented by theempirical Formula (L)_(p)(X)_(q) where L, X, p and q are as definedabove. This is intended to include the several ways in which the ligandscan be linked together in order to achieve the objective ofmultivalency, and a more detailed explanation is described below.

[0342] As noted previously, the linker may be considered as a frameworkto which ligands are attached. Thus, it should be recognized that theligands can be attached at any suitable position on this framework, forexample, at the termini of a linear chain or at any intermediateposition.

[0343] The simplest and most preferred multibinding compound is abivalent compound which can be represented as L—X—L, where each L isindependently a ligand which may be the same or different and each X isindependently the linker. Examples of such bivalent compounds areprovided in FIG. 1 where each shaded circle represents a ligand. Atrivalent compound could also be represented in a linear fashion, i.e.,as a sequence of repeated units L—X—L—X—L, in which L is a ligand and isthe same or different at each occurrence, as can X. However, a trimercan also be a radial multibinding compound comprising three ligandsattached to a central core, and thus represented as (L)₃X, where thelinker X could include, for example, an aryl or cycloalkyl group.Illustrations of trivalent and tetravalent compounds of this inventionare found in FIGS. 2 and 3 respectively where, again, the shaded circlesrepresent ligands. Tetravalent compounds can be represented in a lineararray, e.g.,

L—X—L—X—L—X—L

[0344] in a branched array, e.g.,

[0345] (a branched construct analogous to the isomers of butane—n-butyl,iso-butyl, sec-butyl, and t-butyl) or in a tetrahedral array, e.g.,

[0346] where X and L are as defined herein. Alternatively, it could berepresented as an alkyl, aryl or cycloalkyl derivative as above withfour (4) ligands attached to the core linker.

[0347] The same considerations apply to higher multibinding compounds ofthis invention containing 5-10 ligands as illustrated in FIG. 4 where,as before, the shaded circles represent ligands. However, formultibinding agents attached to a central linker such as aryl orcycloalkyl, there is a self-evident constraint that there must besufficient attachment sites on the linker to accommodate the number ofligands present; for example, a benzene ring could not directlyaccommodate more than 6 ligands, whereas a multi-ring linker (e.g.,biphenyl) could accommodate a larger number of ligands.

[0348] The above described compounds may alternatively be represented ascyclic chains of the form:

[0349] and variants thereof.

[0350] All of the above variations are intended to be within the scopeof the invention defined by the Formula (L)_(p)(X)_(q).

[0351] With the foregoing in mind, a preferred linker may be representedby the following formula:

—X^(a)—Z—(Y^(a)—Z)_(m)—X^(a)—

[0352] wherein

[0353] m is an integer of from 0 to 20;

[0354] X^(a) at each separate occurrence is selected from the groupconsisting of —O—, —S—, —N—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)NR—,—NRC(O)—, C(S), —C(S)O—, —C(S)NR—, —NRC(S)—, or a covalent bond where Ris as defined below;

[0355] Z at each separate occurrence is selected from the groupconsisting of alkylene, substituted alkylene, cycloalkylene, substitutedcylcoalkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, cycloalkenylene, substituted cycloalkenylene,arylene, heteroarylene, heterocyclene, or a covalent bond;

[0356] each Y^(a) at each separate occurrence is selected from the groupconsisting of —O—, —C(O)—, —OC(O)—, —C(O)O—, —NR—, —S(O)_(n)—,—C(O)NR′—, —NR′C(O)—, —NR′C(O)NR′—, —NR′C(S)NR′—, —C(═NR′)—NR′—,—NR′—C(═NR′)—, —OC(O)—NR′—, —NR′—C(O)—O—, —N═C(X^(a))—NR′—,—NR′—C(X^(a))═N—, —P(O)(OR′)—O—, —O—P(O)(OR′)—, —S(O)_(n)CR′R″—,—S(O)_(n)—NR′—, —NR′—S(O)_(n)—, —S—S—, and a covalent bond; where n is0, 1 or 2; and R, R′ and R″ at each separate occurrence are selectedfrom the group consisting of hydrogen, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl,aryl, heteroaryl and heterocyclic.

[0357] Additionally, the linker moiety can be optionally substituted atany atom therein by one or more alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl,substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryland heterocyclic group.

[0358] In view of the above description of the linker, it is understoodthat the term “linker” when used in combination with the term“multibinding compound” includes both a covalently contiguous singlelinker (e.g., L—X—L) and multiple covalently non-contiguous linkers(L—X—L—X—L) within the multibinding compound.

Combinatorial Libraries

[0359] The methods described above lend themselves to combinatorialapproaches for identifying multimeric compounds which possessmultibinding properties.

[0360] Specifically, factors such as the proper juxtaposition of theindividual ligands of a multibinding compound with respect to therelevant array of binding sites on a target or targets is important inoptimizing the interaction of the multibinding compound with itstarget(s) and to maximize the biological advantage through multivalency.One approach is to identify a library of candidate multibindingcompounds with properties spanning the multibinding parameters that arerelevant for a particular target. These parameters include: (1) theidentity of ligand(s), (2) the orientation of ligands, (3) the valencyof the construct, (4) linker length, (5) linker geometry, (6) linkerphysical properties, and (7) linker chemical functional groups.

[0361] Libraries of multimeric compounds potentially possessingmultibinding properties (i.e., candidate multibinding compounds) andcomprising a multiplicity of such variables are prepared and theselibraries are then evaluated via conventional assays corresponding tothe ligand selected and the multibinding parameters desired.Considerations relevant to each of these variables are set forth below:

[0362] Selection of Ligand(s)

[0363] A single ligand or set of ligands is (are) selected forincorporation into the libraries of candidate multibinding compoundswhich library is directed against a particular biological target ortargets e.g., β2 adrenergic receptor. The only requirement for theligands chosen is that they are capable of interacting with the selectedtarget(s). Thus, ligands may be known drugs, modified forms of knowndrugs, substructures of known drugs or substrates of modified forms ofknown drugs (which are competent to interact with the target), or othercompounds. Ligands are preferably chosen based on known favorableproperties that may be projected to be carried over to or amplified inmultibinding forms. Favorable properties include demonstrated safety andefficacy in human patients, appropriate PK/ADME profiles, syntheticaccessibility, and desirable physical properties such as solubility, logP, etc. However, it is crucial to note that ligands which display anunfavorable property from among the previous list may obtain a morefavorable property through the process of multibinding compoundformation; i.e., ligands should not necessarily be excluded on such abasis. For example, a ligand that is not sufficiently potent at aparticular target so as to be efficacious in a human patient may becomehighly potent and efficacious when presented in multibinding form. Aligand that is potent and efficacious but not of utility because of anon-mechanism-related toxic side effect may have increased therapeuticindex (increased potency relative to toxicity) as a multibindingcompound. Compounds that exhibit short in vivo half-lives may haveextended half-lives as multibinding compounds. Physical properties ofligands that limit their usefulness (e.g. poor bioavailability due tolow solubility, hydrophobicity, hydrophilicity) may be rationallymodulated in multibinding forms, providing compounds with physicalproperties consistent with the desired utility.

[0364] Orientation: Selection of Ligand Attachment Points and LinkingChemistry

[0365] Several points are chosen on each ligand at which to attach theligand to the linker. The selected points on the ligand/linker forattachment are functionalized to contain complementary reactivefunctional groups. This permits probing the effects of presenting theligands to their receptor(s) in multiple relative orientations, animportant multibinding design parameter. The only requirement forchoosing attachment points is that attaching to at least one of thesepoints does not abrogate activity of the ligand. Such points forattachment can be identified by structural information when available.For example, inspection of a co-crystal structure of a proteaseinhibitor bound to its target allows one to identify one or more siteswhere linker attachment will not preclude the enzyme:inhibitorinteraction. Alternatively, evaluation of ligand/target binding bynuclear magnetic resonance will permit the identification of sitesnon-essential for ligand/target binding. See, for example, Fesik, etal., U.S. Pat. No. 5,891,643. When such structural information is notavailable, utilization of structure-activity relationships (SAR) forligands will suggest positions where substantial structural variationsare and are not allowed. In the absence of both structural and SARinformation, a library is merely selected with multiple points ofattachment to allow presentation of the ligand in multiple distinctorientations. Subsequent evaluation of this library will indicate whatpositions are suitable for attachment.

[0366] It is important to emphasize that positions of attachment that doabrogate the activity of the monomeric ligand may also be advantageouslyincluded in candidate multibinding compounds in the library providedthat such compounds bear at least one ligand attached in a manner whichdoes not abrogate intrinsic activity. This selection derives from, forexample, heterobivalent interactions within the context of a singletarget molecule. For example, consider a receptor antagonist ligandbound to its target receptor, and then consider modifying this ligand byattaching to it a second copy of the same ligand with a linker whichallows the second ligand to interact with the same receptor molecule atsites proximal to the antagonist binding site, which include elements ofthe receptor that are not part of the formal antagonist binding siteand/or elements of the matrix surrounding the receptor such as themembrane. Here, the most favorable orientation for interaction of thesecond ligand molecule with the receptor/matrix may be achieved byattaching it to the linker at a position which abrogates activity of theligand at the formal antagonist binding site. Another way to considerthis is that the SAR of individual ligands within the context of amultibinding structure is often different from the SAR of those sameligands in momomeric form.

[0367] The foregoing discussion focused on bivalent interactions ofdimeric compounds bearing two copies of the same ligand joined to asingle linker through different attachment points, one of which mayabrogate the binding/activity of the monomeric ligand. It should also beunderstood that bivalent advantage may also be attained withheterodimeric constructs bearing two different ligands that bind tocommon or different targets. For example, a 5HT₄ receptor antagonist anda bladder-selective muscarinic M₃ antagonist may be joined to a linkerthrough attachment points which do not abrogate the binding affinity ofthe monomeric ligands for their respective receptor sites. The dimericcompound may achieve enhanced affinity for both receptors due tofavorable interactions between the 5HT₄ ligand and elements of the M₃receptor proximal to the formal M₃ antagonist binding site and betweenthe M₃ ligand and elements of the 5HT₄ receptor proximal to the formal5HT₄ antagonist binding site. Thus, the dimeric compound may be morepotent and selective antagonist of overactive bladder and a superiortherapy for urinary urge incontinence.

[0368] Once the ligand attachment points have been chosen, oneidentifies the types of chemical linkages that are possible at thosepoints. The most preferred types of chemical linkages are those that arecompatible with the overall structure of the ligand (or protected formsof the ligand) readily and generally formed, stable and intrinsicallyinocuous under typical chemical and physiological conditions, andcompatible with a large number of available linkers. Amide bonds,ethers, amines, carbamates, ureas, and sulfonamides are but a fewexamples of preferred linkages. Linkers: spanning relevant multibindingparameters through selection of valency linker length, linker geometry,rigidity, physical properties, and chemical functional groups

[0369] In the library of linkers employed to generate the library ofcandidate multibinding compounds, the selection of linkers employed inthis library of linkers takes into consideration the following factors:

[0370] Valency

[0371] In most instances the library of linkers is initiated withdivalent linkers. The choice of ligands and proper juxtaposition of twoligands relative to their binding sites permits such molecules toexhibit target binding affinities and specificities more than sufficientto confer biological advantage. Furthermore, divalent linkers orconstructs are also typically of modest size such that they retain thedesirable biodistribution properties of small molecules.

[0372] Linker Length

[0373] Linkers are chosen in a range of lengths to allow the spanning ofa range of inter-ligand distances that encompass the distance preferablefor a given divalent interaction. In some instances the preferreddistance can be estimated rather precisely from high-resolutionstructural information of targets, typically enzymes and solublereceptor targets. In other instances where high-resolution structuralinformation is not available (such as 7TM G-protein coupled receptors),one can make use of simple models to estimate the maximum distancebetween binding sites either on adjacent receptors or at differentlocations on the same receptor. In situations where two binding sitesare present on the same target (or target subunit for multisubunittargets), preferred linker distances are 2-20 Å, with more preferredlinker distances of 3-12 Å. In situations where two binding sites resideon separate (e.g., protein) target sites, preferred linker distances are20-100 Å, with more preferred distances of 30-70 Å.

[0374] Linker Geometry and Rigidity

[0375] The combination of ligand attachment site, linker length, linkergeometry, and linker rigidity determine the possible ways in which theligands of candidate multibinding compounds may be displayed in threedimensions and thereby presented to their binding sites. Linker geometryand rigidity are nominally determined by chemical composition andbonding pattern, which may be controlled and are systematically variedas another spanning function in a multibinding array. For example,linker geometry is varied by attaching two ligands to the ortho, meta,and para positions of a benzene ring, or in cis- or trans-arrangementsat the 1,1- vs. 1,2- vs. 1,3- vs. 1,4-positions around a cyclohexanecore or in cis- or trans-arrangements at a point of ethyleneunsaturation. Linker rigidity is varied by controlling the number andrelative energies of different conformational states possible for thelinker. For example, a divalent compound bearing two ligands joined by1,8-octyl linker has many more degrees of freedom, and is therefore lessrigid than a compound in which the two ligands are attached to the 4,4′positions of a biphenyl linker.

[0376] Tinker Physical Properties

[0377] The physical properties of linkers are nominally determined bythe chemical constitution and bonding patterns of the linker, and linkerphysical properties impact the overall physical properties of thecandidate multibinding compounds in which they are included. A range oflinker compositions is typically selected to provide a range of physicalproperties (hydrophobicity, hydrophilicity, amphiphilicity,polarization, acidity, and basicity) in the candidate multibindingcompounds. The particular choice of linker physical properties is madewithin the context of the physical properties of the ligands they joinand preferably the goal is to generate molecules with favorable PK/ADMEproperties. For example, linkers can be selected to avoid those that aretoo hydrophilic or too hydrophobic to be readily absorbed and/ordistributed in vivo.

[0378] Linker Chemical Functional Groups

[0379] Linker chemical functional groups are selected to be compatiblewith the chemistry chosen to connect linkers to the ligands and toimpart the range of physical properties sufficient to span initialexamination of this parameter.

Combinatorial Synthesis

[0380] Having chosen a set of n ligands (n being determined by the sumof the number of different attachment points for each ligand chosen) andm linkers by the process outlined above, a library of (n!)m candidatedivalent multibinding compounds is prepared which spans the relevantmultibinding design parameters for a particular target. For example, anarray generated from two ligands, one which has two attachment points(A1, A2) and one which has three attachment points (B1, B2, B3) joinedin all possible combinations provide for at least 15 possiblecombinations of multibinding compounds: A1-A1 A1-A2 A1-B1 A1-B2 A1-B3A2-A2 A2-B1 A2-B2 A2-B3 B1-B1 B1-B2 B1-B3 B2-B2 B2-B3 B3-B3

[0381] When each of these combinations is joined by 10 differentlinkers, a library of 150 candidate multibinding compounds results.

[0382] Given the combinatorial nature of the library, common chemistriesare preferably used to join the reactive functionalies on the ligandswith complementary reactive functionalities on the linkers. The librarytherefore lends itself to efficient parallel synthetic methods. Thecombinatorial library can employ solid phase chemistries well known inthe art wherein the ligand and/or linker is attached to a solid support.Alternatively and preferably, the combinatorial libary is prepared inthe solution phase. After synthesis, candidate multibinding compoundsare optionally purified before assaying for activity by, for example,chromatographic methods (e.g., HPLC).

[0383] Analysis of Array by Biochemical, Analytical, Pharmacological,and Computational Methods

[0384] Various methods are used to characterize the properties andactivities of the candidate multibinding compounds in the library todetermine which compounds possess multibinding properties. Physicalconstants such as solubility under various solvent conditions andlogD/clogD values can be determined. A combination of NMR spectroscopyand computational methods is used to determine low-energy conformationsof the candidate multibinding compounds in fluid media. The ability ofthe members of the library to bind to the desired target and othertargets is determined by various standard methods, which includeradioligand displacement assays for receptor and ion channel targets,and kinetic inhibition analysis for many enzyme targets. In vitroefficacy, such as for receptor agonists and antagonists, ion channelblockers, and antimicrobial activity, can also be determined.Pharmacological data, including oral absorption, everted gutpenetration, other pharmacokinetic parameters and efficacy data can bedetermined in appropriate models. In this way, key structure-activityrelationships are obtained for multibinding design parameters which arethen used to direct future work.

[0385] The members of the library which exhibit multibinding properties,as defined herein, can be readily determined by conventional methods.First those members which exhibit multibinding properties are identifiedby conventional methods as described above including conventional assays(both in vitro and in vivo).

[0386] Second, ascertaining the structure of those compounds whichexhibit multibinding properties can be accomplished via art recognizedprocedures. For example, each member of the library can be encrypted ortagged with appropriate information allowing determination of thestructure of relevant members at a later time. See, for example, Dower,et al., International Patent Application Publication No. WO 93/06121;Brenner, et al., Proc. Natl. Acad. Sci., USA, 89:5181 (1992); Gallop, etal., U.S. Pat. No. 5,846,839; each of which are incorporated herein byreference in its entirety. Alternatively, the structure of relevantmultivalent compounds can also be determined from soluble and untaggedlibraries of candidate multivalent compounds by methods known in the artsuch as those described by Hindsgaul, et al., Canadian PatentApplication No. 2,240,325 which was published on Jul. 11, 1998. Suchmethods couple frontal affinity chromatography with mass spectroscopy todetermine both the structure and relative binding affinities ofcandidate multibinding compounds to receptors.

[0387] The process set forth above for dimeric candidate multibindingcompounds can, of course, be extended to trimeric candidate compoundsand higher analogs thereof.

[0388] Follow-up Synthesis and Analysis of Additional Array(s)

[0389] Based on the information obtained through analysis of the initiallibrary, an optional component of the process is to ascertain one ormore promising multibinding “lead” compounds as defined by particularrelative ligand orientations, linker lengths, linker geometries, etc.Additional libraries can then be generated around these leads to providefor further information regarding structure to activity relationships.These arrays typically bear more focused variations in linker structurein an effort to further optimize target affinity and/or activity at thetarget (antagonism, partial agonism, etc.), and/or alter physicalproperties. By iterative redesign/analysis using the novel principles ofmultibinding design along with classical medicinal chemistry,biochemistry, and pharmacology approaches, one is able to prepare andidentify optimal multibinding compounds that exhibit biologicaladvantage towards their targets and as therapeutic agents.

[0390] To further elaborate upon this procedure, suitable divalentlinkers include, by way of example only, those derived from dicarboxylicacids, disulfonylhalides, dialdehydes, diketones, dihalides,diisocyanates,diamines, diols, mixtures of carboxylic acids,sulfonylhalides, aldehydes, ketones, halides, isocyanates, amines anddiols. In each case, the carboxylic acid, sulfonylhalide, aldehyde,ketone, halide, isocyanate, amine and diol functional group is reactedwith a complementary functionality on the ligand to form a covalentlinkage. Such complementary functionality is well known in the art asillustrated in the following table:

Complementary Binding Chemistries

[0391] First Reactive Group Second Reactive Group Linkage hydroxylisocyanate urethane amine epoxide β-hydroxyamine hydroxyamine sulfonylhalide sulfonamide carboxyl acid amine amide hydroxyl alkyl/aryl halideether aldehyde amine/NaCNBH₃ amine ketone amine/NaCNBH₃ amine amineisocyanate urea

[0392] The following table illustrates, by way of examples, startingmaterials (identified as X-1 through X-418) that can be used to preparelinkers incorporated in the multibinding compounds of this inventionutilizing the chemistry described above. For example,1,10-decanedicarboxylic acid, X1, can be reacted with 2 equivalents of aligand carrying an amino group in the presence of a coupling reagentsuch as DCC to provide a bivalent multibinding compound of formula (I)wherein the ligands are linked via 1,10-decanediamido linking group.

[0393] Representative ligands for use in this invention include, by wayof example, L-1 and L-2 as identified above wherein L-1 is selected froma compound of formula (a) and L-2 is selected from a compound of formula(b).

[0394] Combinations of ligands (L) and linkers (X) per this inventioninclude, by way example only, homo- and hetero-dimers wherein a firstligand is selected from L-1 and the second ligand and linker is selectedfrom the following: L-2/X-1- L-2/X-2- L-2/X-3- L-2/X-4- L-2/X-5-L-2/X-6- L-2/X-7- L-2/X-8- L-2/X-9- L-2/X-10- L-2/X-11- L-2/X-12-L-2/X-13- L-2/X-14- L-2/X-15- L-2/X-16- L-2/X-17- L-2/X-18- L-2/X-19-L-2/X-20- L-2/X-21- L-2/X-22- L-2/X-23- L-2/X-24- L-2/X-25- L-2/X-26-L-2/X-27- L-2/X-28- L-2/X-29- L-2/X-30- L-2/X-31- L-2/X-32- L-2/X-33-L-2/X-34- L-2/X-35- L-2/X-36- L-2/X-37- L-2/X-38- L-2/X-39- L-2/X-40-L-2/X-41- L-2/X-42- L-2/X-43- L-2/X-44- L-2/X-45- L-2/X-46- L-2/X-47-L-2/X-48- L-2/X-49- L-2/X-50- L-2/X-51- L-2/X-52- L-2/X-53- L-2/X-54-L-2/X-55- L-2/X-56- L-2/X-57- L-2/X-58- L-2/X-59- L-2/X-60- L-2/X-61-L-2/X-62- L-2/X-63- L-2/X-64- L-2/X-65- L-2/X-66- L-2/X-67- L-2/X-68-L-2/X-69- L-2/X-70- L-2/X-71- L-2/X-72- L-2/X-73- L-2/X-74- L-2/X-75-L-2/X-76- L-2/X-77- L-2/X-78- L-2/X-79- L-2/X-80- L-2/X-81- L-2/X-82-L-2/X-83- L-2/X-84- L-2/X-85- L-2/X-86- L-2/X-87- L-2/X-88- L-2/X-89-L-2/X-90- L-2/X-91- L-2/X-92- L-2/X-93- L-2/X-94- L-2/X-95- L-2/X-96-L-2/X-97- L-2/X-98- L-2/X-99- L-2/X-100- L-2/X-101- L-2/X-102-L-2/X-103- L-2/X-104- L-2/X-105- L-2/X-106- L-2/X-107- L-2/X-108-L-2/X-109- L-2/X-110- L-2/X-111- L-2/X-112- L-2/X-113- L-2/X-114-L-2/X-115- L-2/X-116- L-2/X-117- L-2/X-118- L-2/X-119- L-2/X-120-L-2/X-121- L-2/X-122- L-2/X-123- L-2/X-124- L-2/X-125- L-2/X-126-L-2/X-127- L-2/X-128- L-2/X-129- L-2/X-130- L-2/X-131- L-2/X-132-L-2/X-133- L-2/X-134- L-2/X-135- L-2/X-136- L-2/X-137- L-2/X-138-L-2/X-139- L-2/X-140- L-2/X-141- L-2/X-142- L-2/X-143- L-2/X-144-L-2/X-145- L-2/X-146- L-2/X-147- L-2/X-148- L-2/X-149- L-2/X-150-L-2/X-151- L-2/X-152- L-2/X-153- L-2/X-154- L-2/X-155- L-2/X-156-L-2/X-157- L-2/X-158- L-2/X-159- L-2/X-160- L-2/X-161- L-2/X-162-L-2/X-163- L-2/X-164- L-2/X-165- L-2/X-166- L-2/X-167- L-2/X-168-L-2/X-169- L-2/X-170- L-2/X-171- L-2/X-172- L-2/X-173- L-2/X-174-L-2/X-175- L-2/X-176- L-2/X-177- L-2/X-178- L-2/X-179- L-2/X-180-L-2/X-181- L-2/X-182- L-2/X-183- L-2/X-184- L-2/X-185- L-2/X-186-L-2/X-187- L-2/X-188- L-2/X-189- L-2/X-190- L-2/X-191- L-2/X-192-L-2/X-193- L-2/X-194- L-2/X-195- L-2/X-196- L-2/X-197- L-2/X-198-L-2/X-199- L-2/X-200- L-2/X-201- L-2/X-202- L-2/X-203- L-2/X-204-L-2/X-205- L-2/X-206- L-2/X-207- L-2/X-208- L-2/X-209- L-2/X-210-L-2/X-211- L-2/X-212- L-2/X-213- L-2/X-214- L-2/X-215- L-2/X-216-L-2/X-217- L-2/X-218- L-2/X-219- L-2/X-220- L-2/X-221- L-2/X-222-L-2/X-223- L-2/X-224- L-2/X-225- L-2/X-226- L-2/X-227- L-2/X-228-L-2/X-229- L-2/X-230- L-2/X-231- L-2/X-232- L-2/X-233- L-2/X-234-L-2/X-235- L-2/X-236- L-2/X-237- L-2/X-238- L-2/X-239- L-2/X-240-L-2/X-241- L-2/X-242- L-2/X-243- L-2/X-244- L-2/X-245- L-2/X-246-L-2/X-247- L-2/X-248- L-2/X-249- L-2/X-250- L-2/X-251- L-2/X-252-L-2/X-253- L-2/X-254- L-2/X-255- L-2/X-256- L-2/X-257- L-2/X-258-L-2/X-259- L-2/X-260- L-2/X-261- L-2/X-262- L-2/X-263- L-2/X-264-L-2/X-265- L-2/X-266- L-2/X-267- L-2/X-268- L-2/X-269- L-2/X-270-L-2/X-271- L-2/X-272- L-2/X-273- L-2/X-274- L-2/X-275- L-2/X-276-L-2/X-277- L-2/X-278- L-2/X-279- L-2/X-280- L-2/X-281- L-2/X-282-L-2/X-283- L-2/X-284- L-2/X-285- L-2/X-286- L-2/X-287- L-2/X-288-L-2/X-289- L-2/X-290- L-2/X-291- L-2/X-292- L-2/X-293- L-2/X-294-L-2/X-295- L-2/X-296- L-2/X-297- L-2/X-298- L-2/X-299- L-2/X-300-L-2/X-301- L-2/X-302- L-2/X-303- L-2/X-304- L-2/X-305- L-2/X-306-L-2/X-307- L-2/X-308- L-2/X-309- L-2/X-310- L-2/X-311- L-2/X-312-L-2/X-313- L-2/X-314- L-2/X-315- L-2/X-316- L-2/X-317- L-2/X-318-L-2/X-319- L-2/X-320- L-2/X-321- L-2/X-322- L-2/X-323- L-2/X-324-L-2/X-325- L-2/X-326- L-2/X-327- L-2/X-328- L-2/X-329- L-2/X-330-L-2/X-331- L-2/X-332- L-2/X-333- L-2/X-334- L-2/X-335- L-2/X-336-L-2/X-337- L-2/X-338- L-2/X-339- L-2/X-340- L-2/X-341- L-2/X-342-L-2/X-343- L-2/X-344- L-2/X-345- L-2/X-346- L-2/X-347- L-2/X-348-L-2/X-349- L-2/X-350- L-2/X-351- L-2/X-352- L-2/X-353- L-2/X-354-L-2/X-355- L-2/X-356- L-2/X-357- L-2/X-358- L-2/X-359- L-2/X-360-L-2/X-361- L-2/X-362- L-2/X-363- L-2/X-364- L-2/X-365- L-2/X-366-L-2/X-367- L-2/X-368- L-2/X-369- L-2/X-370- L-2/X-371- L-2/X-372-L-2/X-373- L-2/X-374- L-2/X-375- L-2/X-376- L-2/X-377- L-2/X-378-L-2/X-379- L-2/X-380- L-2/X-381- L-2/X-382- L-2/X-383- L-2/X-384-L-2/X-385- L-2/X-386- L-2/X-387- L-2/X-388- L-2/X-389- L-2/X-390-L-2/X-391- L-2/X-392- L-2/X-393- L-2/X-394- L-2/X-395- L-2/X-396-L-2/X-397- L-2/X-398- L-2/X-399- L-2/X-400- L-2/X-401- L-2/X-402-L-2/X-403- L-2/X-404- L-2/X-405- L-2/X-406- L-2/X-407- L-2/X-408-L-2/X-409- L-2/X-410- L-2/X-411- L-2/X-412- L-2/X-413- L-2/X-414-L-2/X-415- L-2/X-416- L-2/X-417- L-2/X-418-

[0395] and so on.

Utility, Testing, and Administration Utility

[0396] The multibinding compounds of this invention are β2 adrenergicreceptor agonists or partial agonists. Accordingly, the multibindingcompounds and pharmaceutical compositions of this invention are usefulin the treatment and prevention of diseases mediated by β2 adrenergicreceptor such as asthma, chronic obstructive pulmonary disease,bronchitis, and the like. They are also useful in the treatment ofnervous system injury and premature labor. It is also contemplated thatthe compounds of this invention are useful for treating metabolicdisorders such as obesity, diabetes, and the like.

Testing

[0397] The β2 adrenergic receptor agonistic activity of the compounds ofFormula (I) to may be demonstrated by a variety of in vitro assays knownto those of ordinary skill in the art, such as the assay described inthe biological Examples 1 and 2. It may also be assayed by the ex vivoassays described in Ball, D. I. et al., “Salmeterol a Novel, Long-actingbeta 2-Adrenergic Agonist: Characterization of Pharmacological Activityin Vitro and in Vivo” Br. J. Pharmacol., 104, 665-671 (1991); Linden, A.et al., “Salmeterol, Formoterol, and Salbutamol in the IsolatedGuinea-Pig Trachea: Differences in Maximum Relaxant Effect and Potencybut not in Functional Atagonism. Thorax, 48, 547-553, (1993); and Bials,A. T. et al., Investigations into Factors Determining the Duration ofAction of the Beta 2-Adrenoceptor Agonist, Salmeterol. Br. J.Pharmacol., 108, 505-515 (1993); or in vivo assays such as thosedescribed in Ball, D. I. et al., “Salmeterol a Novel, Long-acting beta2-Adrenergic Agonist: Characterization of Pharmacological Activity inVitro and in Vivo” Br. J. Pharmacol., 104, 665-671 (1991); Kikkawa, H.et al., “TA-2005, a Novel, Long-acting, and Selective Beta2-Adrenoceptor Agonist: Characterization of its in vivo BronchodilatingAction in Guinea Pigs and Cats in Comparison with other Beta2-Agonists”. Biol. Pharm. Bull., 17, 1047-1052, (1994); and Anderson, G.P., “Formoterol: Pharmacology, Molecular basis of Agonism and Mechanismof Long Duration of a Highly Potent and Selective Beta 2-AdrenoceptorAgonist Bronchodilator, Life Sciences, 52, 2145-2160, (1993).

Pharmaceutical Formulations

[0398] When employed as pharmaceuticals, the compounds of this inventionare usually administered in the form of pharmaceutical compositions.These compounds can be administered by a variety of routes includingoral, rectal, transdermal, subcutaneous, intravenous, intramuscular, andinhalation (e.g., intranasal or oral inhalation). These compounds areeffective as injectable, inhaled and oral compositions. Suchcompositions are prepared in a manner well known in the pharmaceuticalart and comprise at least one active compound. A preferred manner foradministering compounds of this invention is inhalation. This is aneffective means for delivering a therapeutic agent directly to therespiratory tract for the treatment of diseases such as asthma and othersimilar or related respiratory tract disorders (see U.S. Pat. No.5,607,915).

[0399] This invention also includes pharmaceutical compositions whichcontain, as the active ingredient, one or more of the compoundsdescribed herein associated with pharmaceutically acceptable carriers.In making the compositions of this invention, the active ingredient isusually mixed with an excipient, diluted by an excipient or enclosedwithin such a carrier which can be in the form of a capsule, sachet,paper or other container. When the excipient serves as a diluent, it canbe a solid, semi-solid, or liquid material, which acts as a vehicle,carrier or medium for the active ingredient. Thus, the compositions canbe in the form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solidor in a liquid medium), ointments containing, for example, up to 10% byweight of the active compound, soft and hard gelatin capsules,suppositories, sterile injectable solutions, and sterile packagedpowders.

[0400] In preparing a formulation, it may be necessary to mill theactive compound to provide the appropriate particle size prior tocombining with the other ingredients. If the active compound issubstantially insoluble, it ordinarily is milled to a particle size ofless than 200 mesh. If the active compound is substantially watersoluble, the particle size is normally adjusted by milling to provide asubstantially uniform distribution in the formulation, e.g. about 40mesh.

[0401] Some examples of suitable excipients include lactose, dextrose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,alginates, tragacanth, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, andmethyl cellulose. The formulations can additionally include: lubricatingagents such as talc, magnesium stearate, and mineral oil; wettingagents; emulsifying and suspending agents; preserving agents such asmethyl- and propylhydroxy-benzoates; sweetening agents; and flavoringagents. The compositions of the invention can be formulated so as toprovide quick, sustained or delayed release of the active ingredientafter administration to the patient by employing procedures known in theart.

[0402] The compositions are preferably formulated in a unit dosage form.The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical excipient. Preferably, the compound of Formula(I) above is employed at no more than about 20 weight percent of thepharmaceutical composition, more preferably no more than about 15 weightpercent, with the balance being pharmaceutically inert carrier(s).

[0403] The active compound is effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. Forexample, when the drug is administered via inhalation, each dosagecontains from about 1 μg to about 1000 μg, preferably about 2 μg toabout 500 μg, more preferably about 5 μg to about 100 μg, even morepreferably about 5 μg to about 60 μg, of the active ingredient. It, willbe understood, however, that the amount of the compound actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered and itsrelative activity, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like.Furthermore, the compound of this invention may be administeredprophylactically, for example, a pharmaceutical composition containing acompound of this invention may be administered before the bronchospasmbegins in an asthma attack, to prevent its occurrence or to reduce theextent to which it occurs.

[0404] For preparing solid compositions such as tablets, the principalactive ingredient is mixed with a pharmaceutical excipient to form asolid preformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepre-formulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid pre-fornulation isthen subdivided into unit dosage forms of the type described abovecontaining the active ingredient of the present invention.

[0405] The tablets or pills of the present invention may be coated orotherwise compounded to provide a dosage form affording the advantage ofprolonged action. For example, the tablet or pill can comprise an innerdosage and an outer dosage component, the latter being in the form of anenvelope over the former. The two components can be separated by anenteric layer which serves to resist disintegration in the stomach andpermit the inner component to pass intact into the duodenum or to bedelayed in release. A variety of materials can be used for such entericlayers or coatings, such materials including a number of polymeric acidsand mixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

[0406] The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as corn oil,cottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

[0407] Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders (see U.S. Pat. No. 5,983,956). Theliquid or solid compositions may contain suitable pharmaceuticallyacceptable excipients as described supra. Preferably the compositionsare administered by the oral or nasal respiratory route for local orsystemic effect. Compositions in preferably pharmaceutically acceptablesolvents may be nebulized by use of inert gases. Nebulized solutions maybe inhaled directly from the nebulizing device or the nebulizing devicemay be attached to a face mask tent, or intermittent positive pressurebreathing machine. Solution, suspension, or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner (see U.S. Pat. Nos. 5,919,827and 5,972,919).

[0408] Furthermore, the pharmaceutical compositions containing one ormore compound(s) of this invention can be administered in combinationany other suitable drug, for example, with a suitable steroidalanti-inflammatory drug, e.g., budesonide, flucatisone, beclamethasone,for the treatment of respiratory disorders. When the combination therapyis employed, the pharmaceutical composition containing the compound(s)of this invention and the steroidal anti-inflammatory drug may beadministered simultaneously, sequentially or separately. Each componentused in the combination therapy is employed in an amount sufficient forits intended purpose. For example, the steriodal anti-inflammatory drugsare employed in sufficient amounts to effect reduction in inflammationin vivo. The β-2 adrenergic receptor agonist/partial agonist compoundsof this invention are employed in an amount sufficient to causerelaxation of smooth muscle tissue, for example, in the bronchialsystem.

[0409] Preferably, the dose range for compounds of this invention isfrom about 1 μg to about 1000 μg per dose, more preferably about 2 μg toabout 500 μg, even more preferably about 5 μg to about 100 μg, and stillmore preferably about 5 μg to about 60 μg. The preferred dosage rangefor a steroidal anti-inflammatory drug is from about 50 to 4800 μg andmore preferably about 100 μg to about 1600 μg. Again, the particulardose used will depend on the patient (age, weight, etc.), and theseverity of the disease (mild, moderate, severe). Lastly, apharmaceutical composition containing the two active ingredients canalso be prepared for administering the drugs simultaneously.

EXAMPLES

[0410] The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof

[0411] In the examples below, the following abbreviations have thefollowing meanings. Unless otherwise stated, all temperatures are indegrees Celsius. If an abbreviation is not defined, it has its generallyaccepted meaning. Å = Angstroms cm = centimeter DCC = dicyclohexylcarbodiimide DMF = N,N-dimethylformamide DMSO = dimethylsulfoxide g =gram HPLC = high performance liquid chromatography MEM = minimalessential medium mg = milligram MIC = minimum inhibitory concentrationmin = minute mL = milliliter mm = millimeter mmol = millimol N = normalTHF = tetrahydrofuran μL = microliters μm = microns rt = roomtemperature R_(f = retention faction) NMR = nuclear magnetic resonanceESMS = electrospray mass spectrum ppm = parts per million

Synthetic Examples Example 1

[0412] Synthesis oftrans-1,4-bis{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}cyclohexane(following FIG. 5)

[0413] Step 1

[0414] To a solution of 5-acetylsalicylic acid methyl ester 11 (5.0 g,25.7 mmole) in dimethylsulfoxide (44 mL) was added 48% hydrobromic acid.The resulting mixture was stirred at 55° C. for 24 h, and poured into aslurry of ice-water (200 mL), precipitating a pale yellow solid. Thesolid was filtered, washed with water (200 mL), and dried to giveα,α-dihydroxy-4-hydroxy-3-methoxycarbonyl-acetophenone 12. The productwas re-suspended in ethyl ether (200 mL), filtered and dried to give(3.41 g, 59%) of pure product. R_(f)=0.8 (10% MeOH/CH₂Cl₂).

[0415] H¹-NMR (4/1 CDCl₃/CD₃OD, 299.96 MHz): δ (ppm) 8.73-8.72 (d, 1H),8.28-8.24 (dd, 1H), 7.08-7.05 (d, 1H), 5.82 (s, 1H), 4.01 (s, 3H).

[0416] Step 2

[0417] To a suspension ofα,α-dihydroxy-4-hydroxy-3-methoxycarbonyl-acetophenone 12 (0.3 g, 1.33mmole) in TBF (10 mL) was added a solution oftrans-1,4-diaminocyclohexane (76 mg, 0.66 mmole) in THF (5 mL). Theresulting suspension was stirred for 3 h at ambient temperature undernitrogen atmosphere, at which formation of an imine was completed judgedby TLC analysis. After cooling of the resulting solution at ice bath, anexcess amount of 2M BH₃—Me₂S in hexane (4 mL, 8 mmole) was added to theprevious solution. The resulting mixture was slowly warmed to rt andrefluxed for 4 h under N₂ stream. After cooling the reaction mixture,MeOH (5 mL) was added to quench excess amount of 2M BH₃—Me₂S. Afterstirring for 30 min., the final solution (or cloudy solution) wasevaporated in vacuo, yielding a pale brown solid. The solid was washedwith EtOAc/hexane (1/2; 20 mL), and dried. The crude product wasdissolved in 50% MeCN/H₂O containing 0.5% TFA, and purified byprep-scale high performance liquid chromatography (HPLC) using a lineargradient (5% to 50% MeCN/H₂O over 50 min, 20 mL/min; detection at 254nM). Fractions with UV absorption were analyzed by LC-MS to isolatetrans-1,4-bis{N-[2-(4-hydroxy-3-hydroxymethyl-phenyl)-2-hydroxyethyl]amino}cyclohexane13.

[0418] H¹-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.35 (d, 2H), 7.18 (dd, 2H),6.80-6.78 (d, 2H), 4.88-4.86 (m, 2H), 4.65 (s, 4H), 3.15 (br s, 4H),2.89 (m, 2H), 1.68-1.55 (br m, 4H); ESMS (C₂₄H₃₄N₂O₆): calcd. 446.5,obsd. 447.5 [M+H]⁺.

[0419] Compound 14

[0420] Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with 4,4′-methylenebis(cyclohexylamine)gavebis{4,4′-[N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]cyclohexane}methane.ESMS (C₃₁H₄₆N₂O₆): calcd. 542.7, obsd. 543.6 [M+H]⁺.

[0421] Compound 15

[0422] Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with 1,3-cyclohexanebis(methylamine) gave1,3-bis{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]aminomethyl}cyclohexane.ESMS (C₂₇H₃₈N₂O₆): calcd. 474.6, obsd. 475.3 [M+H]⁺.

[0423] Compound 16

[0424] Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with 1,8-diamino-p-menthane gave1,8-bis{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}-p-menthane.ESMS (C₂₈H₄₂N₂O₆): calcd. 502.6, obsd. 503.3 [M+H]⁺.

[0425] Compound 17

[0426] Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with 1,4-bis(3-aminopropyl)piperazine gave1,4-bis{3-[[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]propyl}piperazine.ESMS (C₂₈H₄₄N₄O₆): calcd. 532.6, obsd. 533.3 [M+H]⁺, 555.0 [M+Na]⁺.

[0427] Compound 18

[0428] Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with p-xylylenediamine gave1,4-bis{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]aminomethyl}benzene.ESMS (C₂₆H₃₂N₂O₆): calcd. 468.5, obsd. 469.3 [M+H]⁺, 492.0 [M+Na]⁺.

[0429] Compound 19

[0430] Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with m-xylylenediamine gave1,3-bis{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]aminomethyl}benzene.ESMS (C₂₆H₃₂N₂O₆): calcd. 468.5, obsd. 469.3 [M+H]⁺, 492.0 [M+Na]⁺.

[0431] Compound 20

[0432] Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with 2-arninobenzylamine gave1-{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]aminomethyl}-2-{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}benzene.ESMS (C₂₅H₃₀N₂O₆): calcd. 454.5, obsd. 455.3 [M+H]⁺.

[0433] Compound 21

[0434] Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with 2-(4-aminophenyl)ethylamine gave1-{2-[N-2-[(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]ethyl}-2-{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]benzene.ESMS (C₂₆H₃₂N₂O₆): calcd. 468.5, obsd. 469.3 [M+H]⁺.

[0435] Compound 22

[0436] Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with 4,4′-oxydianiline gave4,4′-bis{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}phenylether.ESMS (C₃₀H₃₂N₂O₇): calcd. 532.6, obsd. 533.3 [M+H⁺, 556.1 [M+Na]⁺.

[0437] Compound 23

[0438] Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with 2-aminobenzylamine gave1-{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]aminomethyl}4-{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}benzene.ESMS (C₂₅H₃₀N₂O₆): calcd. 454.5, obsd. 455.5 [M+H]⁺, 477.3 [M+Na]⁺.

Example 2 Synthesis of1-{2-[N-2-[(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]ethyl}-4-{N-[2-phenyl-2-hydroxyethyl]amino]benzene(following FIG. 6)

[0439]

[0440] To a suspension ofα,α-dihydroxy-4-hydroxy-3-methoxycarbonyl-acetophenone 12, prepared inExample 1, Step 1 above, (0.3 g, 1.33 mmole) in THF (10 mL) was added asolution of 2-(4-aminophenyl)ethylamine 25 (0.181 g, 1.33 mmol) in THE(5 mL). The resulting suspension was stirred for 3 h at ambienttemperature under nitrogen atmosphere, followed by additionα,α-dihydroxy-acetophenone 24 (0.2 g, 1.32 mmole). The reaction mixturewas stirred for 3 h at RT, at which formation of the imine was completedas judged by TLC analysis. The reaction mixture was cooled in an icebath and an excess amount of 2M BH₃—Me₂S in hexane (9 mL; 18 mmole) wasadded. The resulting mixture was slowly warmed to rt, and refluxed for 4h under N₂ stream. After cooling, MeOH (10 mL) was added to quenchexcess amount of BH₃—Me₂S. After stirring 30 min., at rt, the finalsolution (or cloudy suspension) was evaporated in vacuo, to give a palebrown solid. The solid was washed with EtOAc/hexane (1/2; 20 mL), anddried. The crude product was dissolved in 50% MeCN/H₂O containing 0.5%TFA, and purified by prep-scale high performance liquid chromatography(HPLC) using a linear gradient (5% to 50% MeCN/H₂O over 50 min, 20mL/min; detection at 254 nM). Fractions with UV absorption were analyzedby LC-MS to locate1-{2-[N-2-[(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]-ethyl}-4-{N-[2-phenyl-2-hydroxyethylaminobenzene26. ESMS (C₂₅H₃₀N₂O₄): calcd. 422.5, obsd. 423.3 [M+H]⁺.

[0441] Compound 27

[0442] Proceeding as described above, but substitutingα,α-dihydroxy-4-hydroxy-3-methoxycarbonylacetophenone withα,α-dihydroxyacetophenone gave1-{2-[N-[2-phenyl-2-hydroxyethyl]aminoethyl}-4-[N-(2-phenyl-2-hydroxyethyl)amino]-benxene.ESMS (C₂₄H₂₈N₂O₈): calcd. 376.5, obsd. 377.0 [M+H]⁺.

Example 3 Synthesis of1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]-amino]ethyl}-4-[N-(2-phenyl-2-hydroxyethyl)amino]benzene(following FIG. 7)

[0443]

[0444] Step 1

[0445] To a solution of 4-(2-aminoethyl)aniline 25 (20 g, 147 mmole) inmethanol (250 mL) was added (Boc)₂O (32.4 g, 148 mmole) in methanol (50mL) at rt. After stirring for 24 h, the reaction mixture wasconcentrated to dryness to afford a pale yellow oily residue. The oilymaterial solidified slowly; thus it was dissolved in 5% MeOH/CH₂Cl₂, andsubsequently applied to flash silica column chromatography (3 to 10%MeOH/CH₂Cl₂). After purification, 4-(N-Boc-2-aminoethyl)aniline 28 wasobtained as a pale yellow solid (32.95 g, 95%): R_(f)=0.6 in 10%MeOH/CH₂Cl₂. ¹H-NMR (CD₃OD, 299.96 MH): δ (ppm) 6.96-6.93 (d, 2H),6.69-6.65 (d, 2H), 3.20-3.13 (q, 2H), 2.63-2.58 (t, 2H), 1.41 (s, 9H).

[0446] Step 2

[0447] 4-(N-Boc-2-aminoethyl)aniline 28 (1.25 g, 5.29 mmole) wasdissolved in methanol (30 mL), followed by addition of phenyl glyoxal 24(0.708 g, 5.28 mmole). The reaction mixture was stirred for 1 h at rt,prior to addition of NaCNBH₃ (0.665 g, 10.6 mmole). The final mixturewas stirred for 12 h at rt, concentrated, and purified by flash silicacolumn chromatography (2 to 5% MeOH/CH₂Cl₂) to giveN-(2-phenyl-2-hydroxyethyl)-4-(N-Boc-2-aminoethyl)-aniline as a paleyellow oil (1.71 g, 91%): R_(f)=0.18 in 5% MeOH/CH₂Cl₂. ¹H-NMR (CD₃OD,299.96 MHz): δ (ppm) 7.4-7.25 (m, 5H), 7.0-6.95 (d, 2H), 6.63-6.60 (d,2H), 4.85-4.79 (dd, 1H), 3.3-3.21 (t, 2H), 3.2-3.15 (m, 2H), 2.64-2.5(t, 2H), 1.42 (s, 9H).

[0448] Step 3

[0449] A solution ofN-(2-phenyl-2-hydroxyethyl)-4-(N-Boc-2-aminoethyl)aniline (1.7 g, 4.77mmole) in methylene chloride (10 mL) was cooled in ice bath, and TFA (10mL) was slowly added under a stream of nitrogen gas. The reactionmixture was stirred for 1 h, and concentrated to yield a pale yellowoil. The crude material was purified by reversed phase HPLC (10% to 40%MeCN/H₂O over 50 min; 20 mL/min) to giveN-(2-phenyl-2-hydroxyethyl)-4-(2-aminoethyl)aniline 29 as the TFA salt(1.1 g). ¹H-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.42-7.3 (m, 5H), 7.29-7.25(d, 2H), 7.12-7.0 (d, 2H), 4.85-4.82 (m, 1H), 3.45-3.35 (m, 2H),3.18-3.1 (t, 2H), 2.98-2.94 (t, 2H); ESMS (C₁₆H₂₀N₂O₁): calcd. 256.4,obsd 257.1 [M+H]⁺, 278.8 [M+Na]⁺, 513.4 [2M+H]⁺.

[0450] Step 4

[0451] To a solution ofN-(2-phenyl-2-hydroxyethyl)-4-(2-aminoethyl)aniline trifluoroacetatesalt 29 (1.1 g, 2.3 mmole) in methanol (10 mL) was added 5 M NaOHsolution (0.93 mL). After stirring for 10 min., the solution wasconcentrated to dryness. The residue was dissolved in THF (25 mL), and(α,α-dihydroxy-4-hydroxy-3-methoxy-carbonylacetophenone 12 (0.514 g,2.27 mmole) was added. The reaction mixture was stirred for 12 h at rt,cooled to 0° C., and BH₃/Me₂S (1.14 mL, 10 M) was added under nitrogenatmosphere. The reaction mixture was gradually warmed to rt, stirred for2 h at rt, and refluxed for 4 h. The reaction mixture was cooled andmethanol (10 mL) was added slowly. After stirring for 30 min., at rt,the reaction mixture was concentrated to afford a solid residue, whichwas dissolved in MeOH (20 mL) containing 10% TFA. Evaporation of theorganics yielded a pale yellow oil which was purified by reversed phaseHPLC: 10% to 30% MeCN/H₂O over 50 min; 20 mL/min to give1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]-amino]ethyl}-4-[N-(2-phenyl-2-hydroxyethyl)-amino]benzene30 as the TFA salt (0.65 g). ¹H-NMR (CD₃OD, 299.96 MHz): δ (ppm)7.42-7.3 (m, 6H), 7.28-7.24 (d, 2H), 7.18-7.14 (dd, 1H), 7.1-7.07 (d,2H), 6.80-6.77 (d, 1H), 4.86-4.82 (m, 2H), 4.65 (s, 2H), 3.44-3.34 (m,2H), 3.28-3.22 (m, 2H), 3.20-3.14 (m, 2H), 3.04-2.96 (m, 2H); ESMS(C₂₅H₃₀N₂O₄): calcd. 422.5, obsd. 423.1 [M+H]⁺, 404.7 [M-1H₂O]⁺, 387.1[M-2H₂O]⁺.

Example 4 Synthesis of1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]-aminoethyl}4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]benzene(following FIG. 8)

[0452]

[0453] Step 1

[0454] A solution of 4-(N-Boc-2-aminoethyl)aniline 28 (7.0 g, 29.6mmole) in ethanol (100 mL) and (R)-styreneoxide (3.56 g, 29.6 mmole) wasrefluxed for 24 h. The organics were removed to give a pale yellowsolid. N-(2-phenyl-2-(S)-hydroxyethyl)-4-(N-Boc-2-aminoethyl)aniline wasseparated by flash silica column chromatography: 1/2 EtOAc/hexane to 3/1EtOAc/hexane to 3% MeOH in 3/1 EtOAc/hexane: Rf=0.39 in 3% MeOH/CH₂Cl₂.

[0455] Step 2

[0456] A solution ofN-(2-phenyl-2-(S)-hydroxyethyl)-4-(N-Boc-2-aminoethyl)-aniline (2.5 g,7.0 mmole) in CH₂Cl₂ (15 mL) was cooled in an ice bath under stream ofnitrogen and TFA (15 mL) was slowly added. The reaction mixture wasstirred for 2 h at 0° C. and then concentrated in vacuo. The crudeproduct was dissolved in 20% MeCN/H₂O and purified by preparativereversed phase HPLC (5 to 2% MeCN/H₂O over 50 min; 254 nm; 20 mL/min.),to give N-(2-phenyl-2-(S-hydroxyethyl)-4-(2-aminoethyl)anilinetrifluoroacetate salt 31 as a colorless oil. ¹H-NMR (CD₃OD, 299.96 MHz):δ (ppm); 7.45-7.25 (m, 9H), 4.9 (dd, 1H), 3.55-3.45 (m, 2H), 3.21-3.15(t, 2H), 3.05-2.95 (t, 211) ESMS (C₁₆H₂₀N₂O₁): calcd. 256.4, obsd. 257.1[M+H]⁺, 280.2 [M+Na]⁺.

[0457] Step 3

[0458] To a solution ofN-(2-phenyl-2-(S)-hydroxyethyl)-4-(2-aminoethyl)aniline trifluoroacetate31 (0.144 g, 0.3 mmole) in methanol (10 mL) was added aq. NaOH solution(1.0 M, 0.625 mL). The solution was concentrated to dryness and theresidue was dissolved in anhydrous THF (5 mL).α,α-Dihydroxy-4-hydroxy-3-methoxycarbonylacetophenone 12 (0.067 g, 0.3mmole) was added and the reaction mixture was stirred for 12 h at rt.BH₃—Me₂S (0.2 mL, 2 M) was added at 0° C. and the reaction mixture washeated at 75° C. for 6 h. After cooling the reaction mixture in icebath, MeOH (5 mL) was slowly added to it to quench the reaction, and thereaction mixture was stirred for 30 min., at rt. The organics wereremoved and the residue was dissolved in TFA/MeOH (1/9; 20 mL), andconcentrated. The crude product was dissolved in 20% MeCN/H₂O, andpurified by preparative HPLC: 5 to 20% MeCN/H₂O; 20 mL/min; 254 nm.) togive1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]ethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)-amino]benzene33.

[0459]¹H-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.42-7.29 (m, 8H), 7.22-7.18(d, 2H), 7.17-7.14 (dd, 1E), 6.80-6.77 (d, 1H), 4.9-4.85 (m, 2H), 4.65(s, 2H), 3.5-3.34 (m, 2H), 3.28-3.25 (m, 2H), 3.19-3.14 (m, 2H),3.04-2.98 (m, 2H); ESMS (C₂₅H₃₀N₂O₄): calcd. 422.5, obsd. 423.1 [M+H]⁺,446.1 [M+Na]⁺.

[0460] Proceeding as described in Example 4 above but substituting(R)-styreneoxide with (S)-styreneoxide gave1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]ethyl}-4-[N-(2-phenyl-2-(R)-hydroxyethyl)amino]benzene34.

[0461]¹H-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.42-7.28 (m, 8H), 7.20-7.1(m, 3H), 6.80-6.77 (d, 1H), 4.9-4.85 (m, 2H), 4.65 (s, 2H), 3.45-3.34(m, 2H), 3.28-3.25 (m, 2H), 3.19-3.15 (m, 2H), 3.04-2.98 (m, 2H); ESMS(C₂₅H₃₀N₂O₄): calcd. 422.5, obsd. 423.1 [M+H]⁺, 446.1 [M+Na]⁺.

Example 5 Synthesis of1,6-bis{-(N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]aminohexyloxypropyl]phenoxy}hexane(following FIG. 9)

[0462]

[0463] Step 1

[0464] A solution of 3-(4-hydroxyphenyl)-1-propanol 35 (3.3 g, 21.7mmole) and 1,6-di-iodohexane (3.5 g, 8.88 mmole) in dimethylsulfoxide(40 mL) was degassed and saturated with N₂ gas and potassium carbonate(4.5 g, 32.56 mmole) was added. The reaction mixture was stirred at 80°C. for 18 h under nitrogen atmosphere and then quenched with brine (150mL). The product was extracted with EtOAc (200 mL) and the organicextracts were washed with 0.1 M NaOH and brine, and dried with MgSO₄.The organics were removed in vacuo to give a pale brown solid. The solidwas purified by flash silica column chromatography: 4/1 hexane/EtOAc to5% MeOH in 1/1 hexane/EtOAc to give1,6-bis[4-(3-hydroxypropyl)phenoxy]hexane 36 (R_(f)=0.17 in 1/1hexane/EtOAc) in 65% yield (2.23 g). ¹H-NMR (CD₃OD, 299.96 MHz): δ (ppm)7.08-7.05 (d, 4H), 6.80-6.77 (d, 4H), 3.93-3.89 (t, 4H), 3.56-3.52 (t,4H), 2.64-2.56 (t, 4H), 1.81-1.69 (m, 8H), 1.44-1.21 (m, 4H).

[0465] Step 2

[0466] A solution of 1,6-bis[4-(3-hydroxypropyl)phenoxy]hexane 36 (2.2g, 5.69 mmole) in DMF (10 mL) was added to a solution of DMF (40 mL)containing NaH (0.57 g; 60% dispersion in mineral oil) at 0° C. undernitrogen atmosphere and the reaction mixture was heated at 50° C. After1 h, 6-bromohexanenitrile (2.26 mL, 17 mmole) was added and the reactionmixture was heated at 80° C. for 24 h. The reaction mixture was quenchedwith brine solution (100 mL) and was extracted with EtOAc (250 mL). Theorganic phase was washed with brine, dried with MgSO₄, and evaporated invacuo, to give a pale yellow oil. Purification by flash silica columnchromatography: 4/1 to 1/1 hexane/EtOAc afforded1,6-bis[4(5-cyanopentyloxypropyl)]phenoxy]hexane 37 product (R_(f)=0.6in 1/1 EtOAc/hexane). ¹H-NMR (CDCl₃, 299.96 MHz): δ (ppm) 7.09-7.06 (d,4H), 6.82-6.79 (d, 4H), 3.94-3.90 (t, 4H), 3.42-3.37 (m, 8H), 2.64-2.58(t, 4H), 2.40-2.32 (m, 8H), 1.90-1.26 (m, 24H).

[0467] Step 3

[0468] The 1,6-bis[4-(5-cyanopentyloxypropyl)]phenoxylhexane 37 (0.278g, 0.48 mmole) obtained in Step 2 above was added to a mixture of conc.HCl (10 mL) and AcOH (2 mL) and the reaction mixture was heated at 90°C. After 15 h, the reaction mixture was diluted with brine (50 mL),extracted with EtOAc (100 mL), and dried with MgSO₄. Evaporation of theorganic phase afforded the1,6-bis[4-(5-carboxypentyloxypropyl)]phenoxy]hexane 38 as a pale yellowoily residue, which was used in next step without further purification.¹H-NMR (CDCl₃, 299.96 MHz): δ (ppm) 7.09-7.07 (d, 41H), 6.82-6.79 (d,4H), 3.96-3.92 (t, 4H), 3.42-3.56 (m, 8H), 2.64-2.59 (t, 4H), 2.39-2.32(m, 4H), 1.91-1.40 (m, 24H).

[0469] Step 4

[0470] To a solution of2-hydroxy-2-(4-benzyloxy-3-hydroxymethylphenyl)-ethylamine 39 (0.263 g,0.96 mmole) in DMF (8 mL) was added1,6-bis[4-(5-carboxypentyloxypropyl)phenoxy]hexane (˜0.48 mmole),obtained in Step 3 above, HOBt (0.13 g, 0.96 mmole), DIPEA (0.21 mL,1.20 mmole), and PyBOP (0.502 g, 0.96 mmole). After stirring for 24 h atrt, the reaction mixture was diluted with brine (20 mL) and extractedwith EtOAc (50 mL). The organic layer was washed with 0.1 M NaOH, 0.1 MHCl, and brine, and dried over MgSO₄. The organic solvents were removedin vacuo to give 1,6-bis[4-(5-amidopentyloxypropyl)-phenoxy]hexane as apale yellow oily residue (0.45 g).

[0471] Step 5

[0472] A solution of 1,6-bis[4-(5-amidopentyloxypropyl)-phenoxy]hexane(0.45 g, 0.4 mmole) obtained in Step 4 above, in anhydrous TEF (10 mL)was added to a solution of LiAIH₄ (0.16 g, 4.22 mmole) in anhydrous TEIF(40 mL) at 0° C . The reaction mixture was stirred for 4 h at 80° C.under nitrogen atmosphere and then quenched by with 10% NaOH (1 mL) at0° C. After 30 min., the reaction mixture was filtered and theprecipitate was washed with 10% MeOH in TBF (50 mL). The filtrates werecombined and evaporated in vacuo to give a pale yellow oily residue.Purification by flash silica column chromatography: 5% MeOH/CH₂Cl₂ to 3%PrNH₂ in 10% MeOH/CH₂Cl₂ gave the1,6-bis[4-(6-aminohexyloxypropyl)-phenoxy]hexane. ¹H-NMR (CDCl₃, 299.96MHz): δ (ppm) 7.40-7.25 (m, 12H), 7.22-7.18 (d, 2H), 7.09-7.02 (d, 4H),6.91-6.88 (d, 2H), 6.81-6.75 (d, 4H), 5.01 (s, 4H), 4.8-4.75 (m, 2H),4.70 (s, 4H), 3.96-3.83 (q, 4H), 3.42-3.34 (m, 8H), 2.84-2.64 (m, 8H),2.62-2.56 (t, 4H), 1.84-1.75 (m, 8H), 1.57-1.50 (m, 10H), 1.34-1.23 (m,10H).

[0473] Step 6

[0474] A solution of 1,6-bis[4-(6-aminohexyloxypropyl)-phenoxy]hexane(0.16 g, 0.15 mmole) obtained in Step 5 above, in EtOH (40 mL) washydrogenated under H₂ (1 atm) atmosphere with 10% Pd/C catalyst (100 mg)at rt for 24 h. The catalyst was filtered and the filtrate wasconcentrated to afford crude product as a pale yellow oil. Purificationby reversed phase HPLC: 10 to 50% MeCN/H₂O over 40 min; 20 mL/min; 254nm provides1,6-bis{4(N-[2-(4-hydroxy-3-hydroxymethyl-phenyl)-2-hydroxyethyl]aminohexyloxypropyl]-phenoxy}hexane 40. H¹-NMR (CD₃OD, 299.96 MHz): δ(ppm) 7.35 (d, 2H), 7.18-7.15 (dd, 2H), 7.08-7.05 (d, 4H), 6.82-6.77 (m,6H), 4.65 (s, 4H), 3.96-3.92 (t, 4H), 3.45-3.34 (m, 8H), 3.12-3.01 (m,61), 2.94-2.89 (t, 2H), 2.62-2.57 (t, 4H), 1.86-1.43 (m, 28H); ESMS(C₅₄H₈N₂O₁₀): calcd. 917.1, obsd. 917.5 [M]⁺, 940.8 [M+Na]⁺.

Example 6 Synthesis of1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-(R)-hydroxy-ethyl]aminoethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]phenyl(following FIG. 10)

[0475]

[0476] Step 1

[0477] A mixture of 4-(N-Boc-2-aminoethyl)aniline 28 (10 g, 42.34mmole), benzaldehyde (4.52 mL, 44.47 mmole), and molecular sieves 4A (10g) in toluene (100 mL) was refluxed at 95° C. for 15 h. The reactionmixture was filtered and the filtrate was concentrated in vacuo to givea colorless oil. The oil was dissolved in MeOH (150 mL) and AcOH (0.5mL) and NaCNBH₃ (2.79 g, 44.4 mmole) were added. The reaction mixturewas stirred at 0° C. for 1 h and at rt for 2 h and then concentrated invacuo to give a pale yellow oily residue. Purification by flash silicacolumn chromatography: 1/1 hexane/EtOAc gaveN-benzyl-4-(N-Boc-2-aminoethyl)aniline 41 as colorless oil (11.5 g,83%). R_(f)=0.75 in 1/1 hexane/EtOAc. H¹-NMR (CD₃OD, 299.96 MHz): δ(ppm) 7.38-7.2 (m, 5H), 6.87-6.84 (d, 2H), 6.58-6.55 (d, 2H), 4.27 (s,2H), 3.2-3.15 (m, 2H), 2.6-2.56 (t, 2H), 1.41 (s, 9H); ESMS(C₂₀H₂₆N₂O₂): calcd. 326.4, obsd. 328 [M+H]⁺.

[0478] Step 2

[0479] A mixture of N-benzyl-4-(N-Boc-2-amiaoethyl)aniline 41 (10 g,30.7 mmole) and (R)-styreneoxide (3.51 mL, 30.7 mmole) in ETOH (100 mL)was refluxed for 48 h. A small aliquot of the reaction mixture was takenout for liquid chromatographic analysis, which indicated that thedesired adduct2-[(N-benzyl-4-[2-N-Boc-aminoethyl)anilino]-1-phenylethanol was formedas a minor product along with another regio-isomer2-[(N-benzyl-4-[2-N-Boc-aminoethyl)anilino]-2-phenyl-etanol in a ratioof ˜1/2. Evaporation of the solution afforded thick, pale yellow oil,which was purified by flash silica column chromatography: 4/1 to 2/1hexane/EtOAc. After repeated chromatography,2-[(N-benzyl-4-[2-N-Boc-aminoethyl)anilino]-1-phenyl-ethanol wasobtained as a colorless oil (4.01 g, 29%) (R_(f)=0.76 in 2/1hexane/EtOAc). H¹-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.4-7.1 (m, 10H),7.1-7.06 (d, 2H), 6.68-6.65 (d, 2H), 5.0 (t, 1H), 4.52-4.46 (d, 1H),4.26-4.22 (d, 1H), 3.76-3.68 (dd, 1H), 3.56-3.48 (dd, 1H), 3.22-3.12 (m,2H), 2.68-2.56 (m, 2H), 1.41 (s, 9H); ESMS (C₂₈H₃₄N₂O₃): calcd. 446.6,obsd. 447.1 [M+H]⁺, 893.4 [2M+H]⁺.

[0480] Step 3

[0481] To a solution of2-[(N-benzyl-4-[2-N-Boc-aminoethyl)anilino-1-phenyl-ethanol (4.01 g,8.99 mmole) in CH₂Cl₂ (15 mL) maintained in an ice bath was added TFA(15 mL) under stream of nitrogen atmosphere. After stirring at 0° C. for30 min., the reaction mixture was concentrated in vacuo, yielding a paleyellow oil. Purification by flash silica column chromatography: (½hexane/EtOAc to 5% i-PrNH₂ in ½ hexane/EtOAc) gave2-[(N-benzyl-4-[2-aminoethyl)anilino]-1-phenyl-ethanol 42 as a paleyellow oil from such fractions with R_(f) of 0.2 (5% i-PrNH₂ in ½hexane/EtOAc) in 74% yield (2.29 g). H¹-NMR (CD₃OD, 299.96 MHz): δ (ppm)7.38-7.06 (m, 10H), 7.01-6.98 (d, 2H), 6.71-6.68 (d, 2H), 5.02-4.96 (dd,1H), 4.54-4.48 (d, 1H), 4.29-4.23 (d, 1H), 3.76-3.67 (dd, 1H), 3.58-3.50(dd, 1H), 2.82-2.74 (t, 2H), 2.64-2.59 (t, 2H); ESMS (C₂₃H₂₆N₂O₁):calcd. 346.5, obsd. 346.3[M]⁺,

[0482] Step 4

[0483] A mixture of2-[(N-benzyl-4-[2-aminoethyl)anilino]-1-phenylethanol 42 (2.28 g, 6.59mmole), benzaldehyde (0.74 mL, 7.28 mmole), and molecular sieves 4A (4g) in toluene (40 mL) was heated at 90° C. for 14 h. The reactionmixture was cooled and filtered, and the sieves were rinsed withtoluene. The combined filtrates were concentrated to give an oilyresidue which was washed with hexane, and dried. The residue wasdissolved in MeOH (40 mL) containing AcOH (0.4 mL) and the reactionmixture was cooled in an ice bath. NaCNBH₃ (0.62 g, 9.87 mmole) wasadded and the reaction mixture was stirred for 2 h at rt, and thenconcentrated. The oily residue was dissolved in 60% MeCN/H₂O, andpurified by reversed phase preparative liquid chromatography (40 to 80%MeCN/H₂O over 30 min; 30 mL/min) to give2-[(N-benzyl-4-[2-N-benzylaminoethyl)anilino]-1-phenylethanol as the TFAsalt. The product was treated with alkaline brine solution, andextracted with ether (200 mL). The organic layer was dried with NaSO₄,and concentrated, to give2-[(N-benzyl-4-[2-N-benzylaminoethyl)anilino]-1-phenylethanol 43 as acolorless oil (1.36 g). H¹-NMR (CD₃OD, 299.96MHz): δ (ppm) 7.36-7.06 (m,15H), 6.98-6.95 (d, 2H), 6.69-6.60 (d, 2H), 5.01-4.96 (t, 1H), 4.54-4.47(d, 1H), 4.29-4.24 (d, 1H), 3.73 (s, 2H), 3.72-3.68 (dd, 1H), 3.59-3.54(dd, 1H), 2.80-2.74 (m, 2H), 2.70-2.64 (m, 2H); ESMS (C₃₀H₃₂N₂O₁):calcd. 436.6, obsd. 437.2 [M+H]⁺.

[0484] Step 5

[0485] A concentrated solution of2-[(N-benzyl-4-[2-N-benzylamninoethyl)anilino]-1-phenylethanol (1.36 g,3.12 mmole) and compound (S)-4-benzyloxy-3-methoxycarbonylstyreneoxide44 (0.887 g, 3.12 mmole; ˜95% ee) (prepared as described in R. Hett, R.Stare, P. Helquist, Tet. Lett., 35, 9375-9378, (1994)) in toluene (1 mL)was heated at 105° C. for 72 h under nitrogen atmosphere. The reactionmixture was purified by flash silica column chromatography (2/1hexane/EtOAc to 3% MeOH in 1/1 hexane/EtOAc) to give1-{2-[N-benzyl—N-2-(4-benzyloxy-3-methoxycarbonylphenyl)-2-(R)-hydroxy]ethylaminoethyl}-4-[N-(2-phenyl-2-(S)-hydroxy)ethylamino]benzene45. (R_(f)=0.62 in 3% MeOH in 1/1 hexane/EtOAc) was obtained as a paleyellow foam (2.0 g, 89%).

[0486] H¹-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.67-7.66 (d, 1H), 7.49-7.42(m, 2H), 7.38-7.0 (m, 20H), 6.88-6.85 (d, 2H), 6.65-6.62 (d, 2H), 5.15(s, 2H), 5.05-4.98 (t, 1H), 4.6-4.54 (t, 1H), 4.53-4.46 (d, 1H),4.28-4.22 (d, 1H), 3.84 (s, 3H), 3.72-3.64 (m, 3H), 3.56-3.46 (dd, 1H),2.74-2.56 (m, 6H); ESMS (C₄₇H₄₈N₂O₅): calcd. 720.9, obsd. 721.4 [M+H]⁺,743.3 [M+Na]⁺.

[0487] Step 6

[0488] To a suspension of LiAlH₄ (0.211 g, 5.56 mmole) in THF (40 mL)cooled with ice bath was added1-{2-[N-benzyl-N-2-(4-benzyloxy-3-methoxycarbonylphenyl)-2-(R)-hydroxyethyl]aminoethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]benzene45 (2.0 g, 2.78 mmole) in ThF (10 mL) under nitrogen atmosphere. Thereaction mixture was warmed slowly to rt and the stirring was continuedfor 5 h. The reaction was cooled to 0° C., and 10% NaOH (0.5 mL) wasslowly added. After 30 min., a thick gel formed. The gel was dilutedwith THP (300 mL), filtered, and the solid mass was rinsed with THF (50mL). The filtrates were combined, and-concentrated in vacuo, yielding anoily residue. The residue was purified by flash silica columnchromatography (2/1 hexane/EtOAc to 3% MeOH in 1/1 hexane/EtOAc) to give1-{2-[N-benzyl—N-2-(4-benzyloxy-3-hydroxymethylphenyl)-2-(R)-hydroxyethyl]aninoethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]benzeneas a colorless oil (1.28 g, 67%). H¹-NMR (CD₃OD, 299.96 MHz): δ (ppm)7.4-7.0 (m, 22H), 6.85-6.82 (m, 3H), 6.63-6.60 (d, 2H), 5.02-4.94 (m,3H), 4.66 (s, 2H), 4.59-4.54 (dd, 1H), 4.48-4.4 (d, 1H), 4.24-4.16 (d,11), 3.76-3.7 (d, 1H), 3.69-3.62 (dd, 1H), 3.58-3.52 (d, 1H), 3.50 3.44(dd, 1H), 2.76-2.54 (m, 6H); ESMS (C₄₆H₄₈N₂O₄): calcd. 692.90, obsd.693.5 [M+H]⁺.

[0489] Step 7

[0490] A solution of1-{2-[N-benzyl-N-2-(4-benzyloxy-3-hydroxymethylphenyl)-2-(R)-hydroxyethyl]amino]ethyl}4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]-benzene(1.28 g, 1.85 mmole) in EtOH (80 mL) was hydrogenated under H₂ (1 atm)with 10% Pd/C (0.6 g) for 36 h. After filtration and rinsing of thecatalyst with EtOH (50 mL), the filtrates were combined, and evaporatedin vacuo, yielding pale yellow foam which was dissolved in 10% MeCN/H₂O,and purified by reversed phase preparative liquid chromatography (10 to30% MeCN/H₂O (containing 0.3% TFA) over 50 min; 30 mL/min; 254 nm) togive1-{2-[N-2-(4-hydroxy-3-hydroxymethyl-phenyl)-2-(R)-hydroxyethyl]aminoethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)-amino]benzeneas the TFA salt (0.6 g, 50%). Optical purity of1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-(R)-hydroxyethyl]aminoethyl}4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]benzene46 which was analyzed with capillary electrophoresis by using a chiralmedium, and estimated to be ˜93%.

[0491] H¹-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.42-7.28 (m, 8H), 7.26-7.22(d,2H), 7.18-7.14 (dd, 1H), 6.80-6.77 (d, 1H), 4.88-4.82 (m, 2H), 4.65(s, 2H), 3.5-3.43 (m, 2H), 3.29-3.26 (m, 2H), 3.19-4.14 (m, 21H),3.06-3.0 (m, 2H); ESMS (C₂₅H₃₀N₂O₄): calcd. 422.5, obsd. 423.1 [M+H]⁺,445.4 [M+Na]⁺.

Example 7 Synthesis of1-{6-[N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-[hydroxyethyl]-amino]hexyloxy}-4-{6-[N-[2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]amino]hexyloxypropyl}benzene(following FIG. 11)

[0492]

[0493] Step 1

[0494] A solution of 3-(4hydroxyphenyl)-1-propanol (2.0 g, 13.1 mmole)in DMF (5 mL ) was added to a solution of DMF (35 mL) containing NaH(1.31 g, 60% in mineral oil) at 0° C. under nitrogen atmosphere. Thereaction mixture was slowly warmed to 80° C. After stirring for 1 h at80° C., the reaction mixture was cooled to 0° C., and6-bromohexanenitrile (5.78 g, 32.83 mmole) was added. The final mixturewas re-heated to 80° C., and stirred for 24 h. The reaction mixture wasquenched with saturated NaCl solution (200 mL), and the product wasextracted with EtOAc (300 mL). The organic layer was washed with brinesolution, dried with Na₂SO₄, and evaporated to dryness, yielding a paleyellow solid. Purification of the crude product by flash silica columnchromatography: 4/1 to 1/1 hexane/EtOAc provided6-{3-[4-(5-cyanopentyloxy)phenyl]propoxy}hexanenitrile in 30% yield(1.33 g). R_(f)=0.63 in 1/1 EtOAc/hexane. ¹H-NMR (CDCl₃, 299.96 MHz): δ(ppm) 7.09-7.07 (d, 2H), 6.81-6.78 (d, 2H), 3.96-3.92 (t, 2H), 3.42-3.37(m, 4H), 2.64-2.58 (t, 2H), 2.39-2.32 (m, 4H), 1.87-1.52 (m, 14 H).

[0495] Step 2

[0496] A solution of 6-{3-[4-(5-pentyloxy)phenyl]propoxy}hexanenitrile(1.33 g, 3.88 mmole) in THF (10 mL) was added to a solution of LiAIH₄(0.442 g, 11.65 mmole) in THF (50 mL) at 0° C. under nitrogenatmosphere. The reaction mixture was heated slowly to reflux, andstirred for 2 h. The reaction mixture was cooled to 0{square root} C.,and 10% NaOH solution (5 mL) was slowly added. After 30 min., thereaction mixture was filtered, and the collected solids were washed withTHF (100 mL). The filtrate was concentrated to yield a pale yellow oilwhich was purified by flash silica column chromatography: 5% MeOH/CH₂Cl₂to 3% i-PrNH₂/20% MeOH/CH₂Cl₂ to give6-{3-[4-(6-aminohexyloxy)-phenyl]propoxy}-hexylamine as a colorless oil(0.5 g, 37%) which was converted to the desired compound by proceedingas described in Example 1, step 2 above. The crude product was purifiedby preparatory reversed phase HPLC: 10 to 40% MeCN/H₂O over 40 m; 20mL/min; 254 nm. ESMS (C₃₉H₅₈N₂O₈): calcd. 682.8, obsd. 683.6 [M+H]⁺,797.5 [M+CF₃CO₂H]⁺.

Example 8 Synthesis ofbis{2-{2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxy]ethyamino}-2-hydroxyethoxy}benzene(following FIG. 12)

[0497]

[0498] Step 1

[0499] To a N₂-saturated solution of acetonitrile (300 mL) containingmethyl 5-acetylsalicylate 50 (20 g, 0.1 mole) and benzylbromide (13.5mL, 0.11 mole) was added K₂CO₃ (28.5 g, 0.21 mole). The reaction mixturewas stirred at 90° C. for 5 h. After cooling, the reaction mixture wasfiltered, and the filtrate was concentrated, in vacuo, yielding a whitesolid which was susended in hexane (300 mL), and collected on Buchnerfunnel to give methyl O-benzyl-5-acetylsalicylate 51 as colorless towhite crystals (28.1 g, 96%). R_(f)=0.69 in 1/1 EtOAc/hexane. H¹-NMR(CDCl₃, 299.96 MHz): δ (ppm) 7.8.43-8.42 (d, 1H), 8.1-8.04 (dd, 1H),7.5-7.28 (m, 5H), 7.08-7.04 (d, 1H), 5.27 (s, 2H), 3.93 (s, 3H), 2.58(s, 3H).

[0500] Step 2

[0501] To a solution of methyl O-benzyl-5-acetylsalicylate 51 (14.15 g,0.05 mole) in CHCl₃ (750 mL) was added bromine (2.70 mL, 0.052 mole).The reaction mixture was stirred at rt. While being stirred, thereaction mixture gradually turned from red-brown to colorless. Themixture was stirred for 2 h at rt, and quenched by adding brine solution(300 mL). After shaking the mixture in a separatory Gel, organic layerwas collected, washed with brine, and dried under Na₂SO₄. The organicsolution was concentrated in vacuo, yielding white solid. It was washedwith ether (200 mL). After drying in air, 15 g (83%) of methylO-benzyl-5-(bromoacetyl)-salicylate 52 was obtained. R_(f)=0.76 in 1/1EtOAc/hexane. H¹-NMR (CDCl₃, 299.96 Mz): δ (ppm) 8.48-8.46 (d, 1H),8.14-8.08 (dd, 1H), 7.51-7.3 (m, 5H), 7.12-7.09 (d, 1H), 5.29 (s, 2H),4.42 (s, 2H), 3.94 (s, 3H).

[0502] Step 3

[0503] To a solution of DMF (60 mL) containing methylO-benzyl-5-(bromoacetyl)-salicylate 52 (7.08 g, 0.019 mole) was addedNaN₃ (1.9 g, 0.029 mole). After stirring at rt for 24 h in the dark, themixture was diluted with EtOAc (200 mL), and washed with brine solution(3×200 mL) in a separatory funnel. The organic phase was dried underMgSO₄, and concentrated to afford pale red solid. It was purified byflash silica column chromatography: 10 to 50% EtOAc in hexane. Thedesired product methyl O-benzyl-5-(azidoacetyl)salicylate 53 wasobtained as white crystals (4.7 g, 74%). R_(f)=0.68 in 1/1 EtOAc/hexane.H¹-NMR (CDCl₃, 299.96 MHz): δ (ppm) 8.38-8.36 (d, 1H), 8.08-8.04 (dd,1H), 7.5-7.3 (m, 5H), 7.12-7.09 (d, 1H), 5.29 (s, 2H), 4.53 (s, 2H),3.94 (s, 3H).

[0504] Step 4

[0505] To a gray suspension of LiAlH₄ (2.74 g, 0.072 mole) in THF (400mL) cooled in ice bath was added methylO-benzyl-5-(azidoacetyl)salicylate 53 (4.7 g, 0.014 mole) under nitrogenatmosphere. The reaction mixture was stirred at 0° C. for 1 h, andgradually warmed to rt. After stirring for 16 h at rt, the mixture washeated at 75° C. for 3 h. The reaction mixture was cooled in ice bath,and quenched by slowly adding 10% NaOH (10 mL). After stirring for 1 h,precipitates were filtered, and rinsed with 5% MeOH in THF (200 mL).Filtrates were combined, and concentrated in vacuo, yielding pale yellowoily residue. The crude product was purified by flash silica columnchromatography: 10% MeOH/CH₂Cl₂ to 5% i-PrNH2 in 30% MeOH/CH₂Cl₂ to give2-(4-benzyloxy-3-hydroxymethylphenyl)-2-hydroxyethylamine 39 as a paleyellow solid (2.6 g, 66%). R_(f)=0.63 in 5% i-PrNH2 in 30% MeOH/CH₂Cl₂.H¹-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.46-7.28 (m, 6H), 7.24-7.20 (dd,1H), 7.0-6.96 (d, 1H), 5.11 (s, 2H), 4.70 (s, 2H), 4.65-4.60 (t, 1H),2.83-2.81 (d, 2H); ESMS (C₁₆H₁₉N₁O₃): calcd. 273.3, obsd. 274.7 [M+H]⁺,547.3 [2M+H]⁺.

[0506] Step 5

[0507] To a solution of EtOH (15 mL) containing compound2-(4-benzyloxy-3-hydroxymethylphenyl)-2-hydroxyethylamine 39 (0.3 g, 1.1mmole) was added resorcinol diglycidyl ether (0.122 g, 0.55 mmole)dissolved in EtOH (5 mL). The reaction mixture was refluxed for 20 h.After cooling down to rt, the reaction mixture was degassed withnitrogen and hydrogenated with 10% Pd/C (0.3 g, 10%) under H₂ (1 atm)atmosphere for 24 h. After filtration of the catalyst, the filtrate wasconcentrated to dryness, yielding a colorless oily residue which waspurified by preparatory reversed phase HPLC (10 to 50% MeCN/H₂O over 40min; 20 mL/min; 254 mn) to givebis{2-{2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxy]-ethyamino}-2-hydroxyethoxy}benzene54. ESMS (C₃₀H₄₀N₂O₁₀): calcd. 588.6, obsd. 589.4 [M+H]⁺, 610.7 [M+Na]⁺.

Example 9 Synthesis of1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxy-ethyl]-amino]ethyl}-4-[N-(2-napth-1-yloxymethyl-2-hydroxyethyl)amino]benzene(following FIG. 13)

[0508]

[0509] Step 1

[0510] A solution of EtOH (50 mL) containing4-(N-Boc-2-aminoethyl)aniline 28 (0.4 g, 1.69 mmole) and3-(1-naphthoxy)-1,2-epoxypropane 55 (0.33 g, 1.65 mmole) was refluxedfor 18 h, and concentrated in vacuo to dryness, yielding a pale yellowoil. It was dissolved in 10 mL of CH₂Cl₂, cooled in ice bath, andtreated with TFA (5 mL). After stirring for 2 h at 0° C., the mixturewas evaporated, yielding a pale red oil. It was dissolved in 30% aqueousacetonitrile, and purified by preparatory HPLC: 10 to 30% MeCN/H₂O over30 min; 20 mL/min; 254 nm. The product 56 was obtained as colorless oil(260 mg; TFA salt). H¹-NMR (CD₃OD, 299.96 MHz): d (ppm) 8.88-8.25 (dd,1H), 7.82-7.79 (dd, 1H), 7.51-7.42 (m, 3H1), 7.39-7.38 (d, 1H),7.33-7.30 (d, 2H), 7.25-7.23 (d, 2H), 6.91-6.89 (d, 1H), 4.37-4.31 (m,1H), 4.22-4.19 (m, 2H), 3.69-3.63 (dd, 1H), 3.67-3.54 (dd, 1H),3.17-3.11 (t, 2H), 2.96-2.91 (t, 2H); ESMS (C₂₁H₂₄N₂O₂): calcd. 336.4,obsd. 337.5 [M+H]⁺, 359.6 [M+Na]³⁰ , 673.4 [2M+H]⁺.

[0511] Step 2

[0512] To a solution of compound 56 (0.13 g, 0.023 mmole; TFA salt) in 5mL of MeOH was added 1.0 M NaOH (1.0 M, 0.46 mL). After homogeneousmixing, the solution was evaporated to dryness. The residue wasdissolved in THF (10 mL), followed by addition of glyoxal 12 (52 mg;0.023 mmole). The resulting suspension was stirred for 4 h at ambienttemperature under nitrogen atmosphere. After cooling of the resultingsolution in ice bath, an excess amount of 2M BH₃—Me₂S in THF (3 mL; 6mmole) was added to the previous reaction solution. The resultingmixture was slowly warmed to rt, and refluxed for 4 h under N₂ stream.After cooling of the hot solution, 5 mL of MeOH was added to the cooledmixture to quench the reaction mixture under nitrogen atmosphere. Aftersting 30 min at rt, the final solution was evaporated in vacuo, yieldinga pale brown solid. It was washed with EtOAc/hexane (1/2; 20 mL), anddried. The crude product was dissolved in 50% MeCN/H₂O containing 0.5%TFA, and purified by prep-scale high performance liquid chromatography(HPLC) using a linear gradient (5% to 50% MeCN/H₂O over 50 min, 20mL/min; detection at 254 nM). Fractions with UV absorption were analyzedby LC-MS to locate the desired product1-{2-[N-2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]amino]-ethyl}4-[N-(2-napth-1-yloxymethyl-2-hydroxy-ethyl)amino]benzene57.ESMS (C₃₀H₃₄N₂O₅): calcd. 502.6, obsd. 503.2 [M+H]⁺, 525.6 [M+Na]⁺.

Example 10 Synthesis of1,4,7-tris{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}octane

[0513]

[0514] To a suspension ofα,α-dihydroxy-4-hydroxy-3-methoxycarbonyl-acetophenone 12 (0.45 g, 1.99mmol) in tetrahydrofuran (15 mL) was added a solution of4-(aminomethyl)-1,8-octadiamine (115 mg, 0.66 mmol) in tetrahydrofuran(5 mL). The resulting suspension was stirred for 12 h at ambienttemperature under nitrogen atmosphere. After cooling of the resultingsolution in ice bath an excess amount of 2 M BH₃—Me₂S in hexane (6 mL,12 mmol) was added. The resulting mixture was slowly warmed to rt, andrefluxed for 6 h under nitrogen atmosphere. After cooling, the reactionmixture was quenched with methanol (5 mL). The resulting solution wasstirred at rt for 30 min., and then concentrated in vacuo to give a palebrown solid. The solid was washed with ethyl acetate:hexane mixture(1:2) and then dried. The crude product was dissolved in 50%acetonitrile/water containing 0.5% TFA and purified by HPLC using alinear gradient (5% to 50% MeCN/H₂O over 50 min., 20 mL/min.; detectionat 254 nM). Fractions with UV absorption was analyzed by LC-MS to locatethe desired product. ESMS (C₃₆H₅₃N₃O₉): Calcd. 671.8; Obsd. 671.7.

Example 11 Synthesis of1-{2-[N-2-(4-amino-3,5-dichlorophenyl)-2-(RS)-hydroxyethyl]aminoethyl}-4-[N-(2-phenyl-2-(RS)-hydroxyethyl)amino]phenyl(following FIG. 14)

[0515]

[0516] Step 1

[0517] A solution of 2-(4-aminophenyl)ethylamine 25 (4.70 mL, 36.7mmol), benzaldehyde (7.46 mL, 73.4 mmol) and 4A molecular sieves (18 g)in toluene (180 ml) was refluxed for 3 h. The reaction mixture wascooled and filtered. The filtrate was concentrated under reducedpressure to afford compound 58 (95% yield).

[0518] To a cooled a solution of 58 (2.00 g, 6.40 mmol) in ethanol (150mL) in ice bath was slowly added sodium borohydride (361 mg, 9.50 mmol)under a nitrogen atmosphere. The reaction mixture was allowed to stir at0° C. for 1.5 h, and then warmed slowly to room temperature. Thereaction mixture was quenched by slowly adding 50% methanol/TFA (5 mL)and then the mixture was concentrated under reduced pressure. Theresultant residue was dissolved in EtOAc, and washed with 0.1 M NaOH.After drying over Na₂SO₄, the organic phase was concentrated in vacuo,and the residue was purified by flash silica gel chromatography usingethyl acetatehexanes as eluant to give compound 59(80% yield).

[0519] Step 3

[0520] To a solution of 59 (1.00 g, 3.20 mmol) in methanol cooled withice bath was slowly added di-tert-butyl dicarbonate (0.69 g, 3.2 mmol)in cold methanol (5 mL). The reaction mixture was allowed to stir at 0°C. for 0.5 h, and then warmed gradually to room temperature. Afterstirring for 1 h, the reaction mixture was concentrated under reducedpressure, and dried under high vacuum overnight. The oily residue waspurified by silica gel chromatography using ethyl acetate/hexane (2:1)to afford compound 60 (80% yield).

[0521] Step 4

[0522] To a solution of 60 (2.09 g, 5.00 mmol) in methanol (45 mL) wasadded phenylglyoxal (2.01 g, 15.0 mmol). The reaction mixture wasstirred at room temperature for 1 h, and followed by slow addition ofsodium cyanoborohydride (1.25 g, 20 mmol). The reaction mixture wasstirred at room temperature overnight. The reaction mixture wasconcentrated under reduced pressure, and the residue was dissolved inmethanol. After filtration, the filtrate was concentrated under reducedpressure. The residue was purified by silica gel chromatography usinghexane/ethyl acetate (9:1) to afford compound 61 (26% yield).

[0523] Step 5

[0524] To a cooled a solution of 61 (3.19 g, 5.90 mmol) in methylenechloride (12 mL) in an ice bath was added slowly trifluoroacetic acid(12 mL) under stream of nitrogen. After stirring the reaction mixturefor 1 h at the same temperature, the mixture was concentrated underreduced pressure to yield compound 62 as an oily residue. The productwas dried in vacuo overnight, and was used in next step without furtherpurification (90% yield).

[0525] Step 6

[0526] To a mixture of 62 (1.87 g, 3.4 mmol) and2,6-dichloro-4-4(bromoacetyl)-aniline 63 (1.07 g, 3.8 mmol) in DMF (50mL) was added potassium carbonate (0.96 g, 6.90 mmol). The reactionmixture was stirred at room temperature for 0.5 h and then at 35° C. for1 h. The reaction mixture was cooled to room temperature, and sodiumborohydride (0.16 g, 4.10 mmol) in ethanol (50 mL) was added slowly. Thereaction continued overnight at room temperature and then quenched withaqueous NH₄Cl (sat'd). After concentration of the reaction mixture underreduced pressure, the residue was dissolved in ether, and washed withbrine. After drying over MgSO₄, the organic layer was concentrated, andthe residue was purified by silica gel chromatography by eluting withhexane/ethyl acetate (4:1). The product 64 was obtained in 50% yield.

[0527] Step 7

[0528] A suspension of 64 (107 mg, 0.17 mmol) and palladium hydroxide(25 mg) in ethanol (2.5 mL) was stirred overnight under hydrogenatmosphere (1 atm) at room temperature. The mixture was filtered, andthe filtrate was concentrated to yield crude product which was purifiedusing silica gel chromatography with 6% methanol/dichloromethane (10%yield) to give compound 65. ESMS (C₂₄H₂₇Cl₂N₃O₂: calcd: 460.4; obsd: 460[M+H]⁺, 442 [M-H₂O]³⁰ , 921 [2M+H]⁺.

Example 12 Synthesis of1-{2-[N-2-(4-hydroxy-3-formylaminophenyl)-2-(RS)-hydroxyethyl]aminoethyl}-4-[N-(2-phenyl-2-(RS)-hydroxyethyl)amino]phenyl(following FIG. 15)

[0529]

[0530] Step 1

[0531] 1-Benzyioxy-4-bromoacetyl-2-nitrobenzene 66 (3.76 g, 10.8 mmol)[prepared as described in Chem. Bull., 25, 1368-1377, (1977)], was addedto a solution of compound 59 (3.4 g, 10.8 mmol) in dimethylformamide(150 mL) at room temperature. After 28 h, the reaction mixture wasdiluted with ether and washed with a dilute solution of aqueous sodiumchloride. The organic layer was separated and dried over sodium sulfate,filtered and concentrated to give a crude oil. Purification with columnchromatography using hexane:ethyl acetate as the eluent providedcompound 67 (95% yield).

[0532] Step 2

[0533] To a solution of 67 (4.0 g, 6.80 mmol) in ethanol (500 mL) wasslowly added sodium borohydride (1 g, 26.50 mmol) in portions over 30min., under a nitrogen atmosphere. After 6 h, the reaction mixture wasquenched by slowly adding saturated solution of aqueous ammoniumchloride. The solution was diluted with 1 N sodium hydroxide and ethylacetate and hexanes. The organic layer was separated, dried over sodiumsulfate, filtered and concentrated to give compound 68 as an oil whichwas used in the next step without further purification.

[0534] Step 3

[0535] To a mixture of 68 (3.6 g, 6.1 mmol) and potassium carbonate (3.0g, 9.2 mmol) in dimethylformamide (100 mL) was addedalpha-bromoacetophenone (1.28 g, 6.4 mmol) portionwise. The reactionmixture was heated overnight at 65° C. An additional portion ofalpha-bromoacetophenone (0.25 g, 1.25 mmol) was added and heating wascontinued. After 18 h, the reaction mixture was cooled to roomtemperature and ethanol (50 mL) was added. Sodium borohydride (1.0 g,26.5 mmol) was added and stirring was continued for 2.5 h. The reactionmixture was concentrated and then methanol (25 mL) was added and theexcess hydride was quenched with the addition of a saturated solution ofammonium chloride. The reaction mixture was diluted with ethyl acetateand ether. The organic layer was separated, dried over sodium sulfate,filtered, and concentrated. The crude oil was purified with columnchromatography eluting with ethyl acetate/hexanes mixture to givecompound 69.

[0536] Step 4

[0537] To a solution of 69 (0.73 g, 1.0 mmol) in a mixture of methanol(20 mL), 6 N hydrochloride acid (1 mL) and water (2 mL) was added ironpowder (0.56 g, 10.0 mmol). The reaction mixture was heated at 90° C.for 1.5 h. The reaction mixture was cooled to room temperature andallowed to stand overnight. Methanol was added and the brownprecipitates and unreacted iron was filtered off. The filtrate wasconcentrated under reduced pressure, to give 70 as a brown solid whichwas used in the next step without further purification.

[0538] Step 5

[0539] Compound 70 was dissolved in a premixed solution of aceticanhydride (5 mL) and formic acid (3 mL) at room temperature. After 3 h,the reaction mixture was diluted with ethyl acetate and evaporated todryness. A methanolic solution of 0.5 M sodium hydroxide was added andthe reaction mixture was stirred for 6 h at room temperature. Thereaction mixture was treated with methanolic solution of 1 Nhydrochloric acid and then concentrated to dryness. The residue wasredissolved in methanol and filtered. The filtrate was concentrated togive compound 71 as a brown residue which was used in the next stepwithout further purification.

[0540] Step 6

[0541] Palladium on carbon (10%, 0.5 g) was added to a suspension of 71in methanol (120 mL) and dimethylformamide (80 mL). The reaction mixturewas purged with nitrogen gas and stirred overnight under hydrogenatmosphere (1 atm) at room temperature. The mixture was filtered, andthe filtrate was concentrated to yield crude product which was purifiedby HPLC (acetonitril/water/1% TFA gradient) to give crude product whichwas purified with column chromatography using methanol/methylenechloride/1%isopropylamine as the eluent to give desired compound1-{2-[N-2-(4-hydroxy-3-formylaminophenyl)-2-(RS)-hydroxyethyl]-aminoethyl}-4[N-(2-phenyl-2-(RS)-hydroxyethyl)amino]phenyl72.

Formulation Examples Example 1

[0542] Hard gelatin capsules containing the following ingredients areprepared: Quantity Ingredient (mg/capsule) Active Ingredient 30.0 Starch305.0 Magnesium stearate 5.0

[0543] The above ingredients are mixed and filled into hard gelatincapsules in 340 mg quantities.

Example 2

[0544] A tablet Formula is prepared using the ingredients below:Quantity Ingredient (mg/tablet) Active Ingredient 25.0 Cellulose,microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0

[0545] The components are blended and compressed to form tablets, eachweighing 240 mg.

Example 3

[0546] A dry powder inhaler formulation is prepared containing thefollowing components: Ingredient Weight Active Ingredient 5 Lactose 95

[0547] The active ingredient is mixed with the lactose and the mixtureis added to a dry powder inhaling appliance.

Example 4

[0548] Tablets, each containing 30 mg of active ingredient, are preparedas follows: Quantity Ingredient (mg/tablet) Active Ingredient  30.0 mgStarch  45.0 mg Microcrystalline cellulose  35.0 mg Polyvinylpyrrolidone(as 10% solution in sterile water)  4.0 mg Sodium carboxymethyl starch 4.5 mg Magnesium stearate  0.5 mg Talc  1.0 mg Total 120.0 mg

[0549] The active ingredient, starch and cellulose are passed through aNo. 20 mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders, which are thenpassed through a 16 mesh U.S. sieve. The granules so produced are driedat 50° to 60° C. and passed through a 16 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 30 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 120 mg.

Example 5

[0550] Capsules, each containing 40 mg of medicament are made asfollows: Quantity Ingredient (mg/capsule) Active Ingredient  40.0 mgStarch 109.0 mg Magnesium stearate  1.0 mg Total 150.0 mg

[0551] The active ingredient, starch, and magnesium stearate areblended, passed through a No. 20 mesh U.S. sieve, and filled into hardgelatin capsules in 150 mg quantities.

Example 6

[0552] Suppositories, each containing 25 mg of active ingredient aremade as follows: Ingredient Amount Active Ingredient   25 mg Saturatedfatty acid glycerides to 2,000 mg

[0553] The active ingredient is passed through a No. 60 mesh U.S. sieveand suspended in the saturated fatty acid glycerides previously meltedusing the minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2.0 g capacity and allowed to cool.

Example 7

[0554] Suspensions, each containing 50 mg of medicament per 5.0 mL doseare made as follows: Ingredient Amount Active Ingredient 50.0 mg Xanthangum  4.0 mg Sodium carboxymethyl cellulose (11%) Microcrystallinecellulose (89%) 50.0 mg Sucrose 1.75 g Sodium benzoate 10.0 mg Flavorand Color    q.v. Purified water to  5.0 mL

[0555] The active ingredient, sucrose and xanthan gum are blended,passed through a No. 10 mesh U.S. sieve, and then mixed with apreviously made solution of the microcrystalline cellulose and sodiumcarboxymethyl cellulose in water. The sodium benzoate, flavor, and colorare diluted with some of the water and added with siring. Sufficientwater is then added to produce the required volume.

Example 8

[0556] A formulation may be prepared as follows: Quantity (mg/capsule)Active Ingredient  15.0 mg Starch 407.0 mg Magnesium stearate  3.0 mgTotal 425.0 mg

[0557] The active ingredient, starch, and magnesium stearate areblended, passed through a No. 20 mesh U.S. sieve, and filled into hardgelatin capsules in 425.0 mg quantities.

Example 9

[0558] A formulation may be prepared as follows: Ingredient QuantityActive Ingredient 5.0 mg Corn Oil 1.0 mL

Example 10

[0559] A topical formulation may be prepared as follows: IngredientQuantity Active Ingredient 1-10 g Emulsifying Wax 30 g Liquid Paraffin20 g White Soft Paraffin to 100 g

[0560] The white soft paraffin is heated until molten. The liquidparaffin and emulsifying wax are incorporated and stirred untildissolved. The active ingredient is added and stirring is continueduntil dispersed. The mixture is then cooled until solid.

[0561] Another preferred formulation employed in the methods of thepresent invention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, e.g.,U.S. Pat. No. 5,023,252, issued Jun. 11, 1991, herein incorporated byreference in its entirety. Such patches may be constructed forcontinuous, pulsatile, or on demand delivery of pharmaceutical agents.

[0562] Other suitable formulations for use in the present invention canbe found in Remington's Pharmaceutical Sciences, edited by E. W. Martin(Mack Publishing Company, 18th ed., 1990).

Biological Examples Example 1 β2-Adrenergic Receptor In Vitro FunctionalAssay

[0563] The β2-adrenergic receptor functional activity of compounds ofthe invention was tested follows.

[0564] Cell Seeding and Growth

[0565] Primary bronchial smooth muscle cells from a 21 yr. old male(Clonetics, San Diego, Calif.) were seeded at 50,000 cells/well in24-well tissue culture plates. The media used was Clonetic's SmBM-2supplemented with hEGF, Insulin, hFGF, and Fetal Bovine Serum. Cellswere grown two days at 37° C., 5% CO₂ until confluent monolayers wereseen.

[0566] Agonist Stimulation of Cells

[0567] The media was aspirated from each well and replaced with 250 mlfresh media containing 1 mM IBMX, a phospodiesterase inhibitor (Sigma,St Louis, Mo.). Cells were incubated for 15 minutes at 37° C., and then250 ml of agonist at appropriate concentration was added. Cells werethen incubated for an additional 10 minutes. Media was aspirated and 500ml cold 70% EtOH was added to cells, and then removed to an empty96-well deep-well plate after about 5 minutes. This step was thenrepeated. The deep-well plate was then spun in a speed-vac until allEtOH dried off, leaving dry pellets. cAMP (pmol/well) was quantitatedusing a cAMP ELISA kit from Stratagene (La Jolla, Calif.). EC₅₀ curveswere generated using the 4-parameter fit equation:

[0568] y=(a-d)/(1+(x/c)^(b))+d, where,

[0569] y=cpm a=total binding c=IC₅₀

[0570] x=[compound] d=NS binding b=slope

[0571] Fix NS binding and allow all other parameters to float.

Example 2 β2-Adrenergic Receptor In Vitro Radioligand Binding Assay

[0572] The β1/2-adrenergic receptor binding activity of compounds of theinvention can be tested follows. SF9 cell membranes containing either β1or β2-adrenergic receptor (NEN, Boston, Mass.) were incubated with 0.07nM ¹²⁵I-iodocyanopindolol (NEN, Boston, Mass.) in binding buffercontaining 75 mM Tris-HCl (pH 7.4), 12.5 mM MgCl₂ and 2 mM EDTA andvarying concentrations of test compounds or buffer only (control) in96-well plates. The plates were incubated at room temperature withshaking for 1 hour. The receptor bound radioligand was harvested byfiltration over 96-well GF/B filter plates (Packard, Meriden, Conn.)pre-blocked with 0.3% polyethylenimine and washed twice with 200 μl PBSusing cell harvester. The filters were washed thrice with 200 μl PBSusing cell harvester and then resuspended in 40 μl scintillationcocktail. The filter-bound radioactivity was measured with ascintillation counter and IC₅₀ curves are generated using the standard4-parameter fit equation described above.

[0573] The foregoing invention has been described in some detail by wayof illustration and example, for purposes of clarity and understanding.It will be obvious to one of skill in the art that changes andmodifications may be practiced within the scope of the appended claims.Therefore, it is to be understood that the above description is intendedto be illustrative and not restrictive. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to thefollowing appended claims, along with the full scope of equivalents towhich such claims are entitled.

[0574] All patents, patent applications and publications cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each individual patent, patentapplication or publication were so individually denoted.

What is claimed is:
 1. A multibinding compound of Formula (I):(L)_(p)(X)_(q)  (I) wherein: p is an integer of from 2 to 10; q is aninteger of from 1 to 20; X is a linker; and L is a ligand wherein: oneof the ligands, L, is a compound of formula (a):

wherein: Ar¹ and Ar² are independently selected from the groupconsisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, andheterocyclyl wherein each of said Ar¹ and Ar² substituent optionallylinks the ligand to a linker; R¹ is selected from the group consistingof hydrogen, alkyl, and substituted alkyl, or R¹ is a covalent bondlinking the ligand to a linker; R² is selected from the group consistingof hydrogen, alkyl, aralkyl, acyl, substituted alkyl, cycloalkyl, andsubstituted cycloalkyl, or R² is a covalent bond linking the ligand to alinker; W is a covalent bond linking the —NR²— group to Ar², alkylene orsubstituted alkylene wherein one or more of the carbon atoms in saidalkylene or substituted alkylene group is optionally replaced by asubstituent selected from the group consisting of —NR^(a)— (where R^(a)is hydrogen, alkyl, acyl, or a covalent bond linking the ligand to alinker), —O—, —S(O)_(n) (where n is an integer of from 0 to 2), —CO—,—PR^(b)— (where R^(b) is alkyl), —P(O)₂—, and —O—P(O)O— and furtherwherein said alkylene or substituted alkylene group optionally links theligand to a linker provided that at least one of Ar¹, Ar², R¹, R², or Wlinks the ligand to a linker; and the other ligands are independently ofeach other a compound of formula (b): —Q—Ar³  (b) wherein: Ar³ isselected from the group consisting of aryl, heteroaryl, cycloalkyl,substituted cycloalkyl, and heterocyclyl; Q, which links the otherligand to the linker, is selected from the group consisting of acovalent bond, alkylene, and substituted alkylene wherein one or more ofthe carbon atoms in said alkylene or substituted alkylene is optionallyreplaced by a substituent selected from —NR^(a)— (where R^(a) ishydrogen, alkyl, acyl, or a covalent bond linking the ligand to alinker), —O—, —S(O)_(n)— (where n is an integer of from 0 to 2), —CO—,—PR^(b)— (where R^(b) is alkyl), —P(O)₂—, and —O—P(O)O—; and individualisomers, mixtures of isomers and pharmaceutically acceptable saltsthereof provided that: (i) when the multibinding compound of Formula (I)is a compound of formula:

 where Ar¹ and Ar³ are aryl, then W and X both are not alkylene oralkylene-O—; (ii) when the multibinding compound of Formula (I) is acompound of formula:

 where Ar¹ is 4-hydroxy-2-methylphenyl, Ar² is aryl, Ar³ is aryl orheterocyclyl, W is ethylene, Q is a covalent bond, R¹ is alkyl, then thelinker X is not linked to the Ar² group through an oxygen atom; (iii)when the multibinding compound of Formula (I) is a compound of formula:

 where Ar¹, Ar², Ar³, R¹, R² are as defined above, W is alkylene, and Qis a covalent bond, then X is not -alkylene-O—; and (iv) when themultibinding compound of Formula (I) is a compound of formula:

 where Ar¹ is 4-benzyloxy-3-formylamino, R² is aralkyl, W is—CH(CH₃)CH₂—, Ar² and Ar³ are phenyl, Q is a covalent bond, then thelinker X is not linked to the Ar² group through an oxygen atom.
 2. Themultibinding compound of claim 2 wherein q is less than p.
 3. Themultibinding compound of claim 2 wherein each linker, X, in themultibinding compound of Formula (I) independently has the formula:—X^(a)—Z—(Y^(a)—Z)_(m)—X^(a)— wherein m is an integer of from 0 to 20;X^(a) at each separate occurrence is selected from the group consistingof —O—, —S—, —NR—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)NR—, —NRC(O)—, C(S),—C(S)O—, —C(S)NR—, —NRC(S)—, or a covalent bond where R is as definedbelow; Z at each separate occurrence is selected from the groupconsisting of alkylene, substituted alkylene, cycloalkylene, substitutedcycloalkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, cycloalkenylene, substituted cycloalkenylene,arylene, heteroarylene, heterocyclene, or a covalent bond; each Y^(a) ateach separate occurrence is selected from the group consisting of —O—,—C(O)—, —OC(O)—, —C(O)O—, —NR—, —S(O)_(n)—, —C(O)NR′—, —NR′C(O)—,—NR′C(O)NR′—, —NR′C(S)NR′—, —C(═NR′)—NR′—, —NR′—C(═NR′)—, —OC(O)—NR′—,—NR′—C(O)—O—, —N═C(X^(a))—NR′—, —NR′—C(X^(a))═N—, —P(O)(OR′)—O—,—O—P(O)(OR′)—, —S(O)_(n)CR′R″—, —S(O)_(n)—NR′—, —NR′—S(O)_(n)—, —S—S—,and a covalent bond; where n is 0, 1 or 2; R, R′ and R″ at each separateoccurrence are selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, aryl, heteroaryl and heterocyclic, and X^(a) is asdefined above.
 4. A multibinding compound of Formula (II):

wherein: Ar¹ is: (a) a phenyl ring of formula (c):

wherein: R⁴ is hydrogen, alkyl, halo, or alkoxy; R⁵ is hydrogen,hydroxy, halo, amino, or —NHSO₂R^(a) where R^(a) is alkyl; R⁶ ishydrogen, halo, hydroxy, alkoxy, substituted alkyl, sulfonylamino,aminoacyl, or acylamino; W is a covalent bond linking the —NR²— group toAr², an alkylene or substituted alkylene group wherein one or more ofthe carbon atoms in the alkylene or substituted alkylene group areoptionally replaced by —O—; A ² is phenyl wherein the W and the X groupsare attached at the 1,2-, 1,3, and 1,4 positions of the phenyl ring;cyclohexyl optionally substituted with methyl and wherein the W and theX groups are attached at the 1,3, and 1,4 positions of the cyclohexylring, or piperazine wherein the W and the X groups are attached at the1,4 positions of the piperazine ring; X is a compound of formula:—X^(a)—Z—(Y^(a)—Z)_(m)—X^(a)— wherein m is an integer of from 0 to 20;X^(a) at each separate occurrence is selected from the group consistingof —O—, —S—, —NR—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)NR—, —NRC(O)—, C(S),—C(S)O—, —C(S)NR—, —NRC(S)—, or a covalent bond where R is as definedbelow; Z at each separate occurrence is selected from the groupconsisting of alkylene, substituted alkylene, cycloalkylene, substitutedcycloalkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, cycloalkenylene, substituted cycloalkenylene,arylene, heteroarylene, heterocyclene, or a covalent bond; each Y^(a) ateach separate occurrence is selected from the group consisting of —O—,—C(O)—, —OC(O)—, —C(O)O—, —NR—, —S(O)_(n)—, —C(O)NR′—, —NR′C(O)—,—NR′C(O)NR′—, —NR′C(S)NR′—, —C(═NR′)—NR′—, —NR′—C(═NR′)—, —OC(O)—NR′—,—NR′—C(O)—O—, —N═C(X^(a))—NR′—, —NR′—C(X^(a))═N—, —P(O)(OR′)—O—,—O—P(O)(OR′)—, —S(O)_(n)CR′R″—, —S(O)_(n)—NR′—, —NR′—S(O)_(n)—, —S—S—,and a covalent bond; where n is 0, 1 or 2; R, R′ and R″ at each separateoccurrence are selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, aryl, heteroaryl and heterocyclic, and X^(a) is asdefined above; Q is a covalent bond, alkylene, or a substituted alkylenegroup wherein one or more of the carbon atoms in said alkylene orsubstituted alkylene group is optionally replaced by a heteroatomselected from the group consisting of —NR^(a)— (where R^(a) is hydrogen,alkyl, acyl, or a covalent bond linking the ligand to a linker), —O—,—S(O)_(n) (where n is an integer of from 0 to 2), —CO—, —PR^(b)— (whereR^(b) is alkyl), —P(O)₂—, and —O—P(O)O— and links the ligand to alinker; and Ar³ is either: (i) a phenyl ring of formula (c) as definedabove; or (ii) a phenyl ring of formula (d):

wherein: R⁷ is hydrogen, alkyl, alkenyl, substituted alkyl, halo,alkoxy, substituted alkoxy, hydroxy, aminoacyl, or heteroaryl; and R⁸ ishydrogen, halo, alkoxy, substituted alkoxy, or acylamino; or (iii)naphthyl, pyridyl, benzimidazol-1-yl, indolyl, 2-cyanoindolyl,carbazolyl, 4-methylindanyl, 5-(CH₃CO₂CH₂O—)-1,2,3,4-tetrahydronaphthyl,1H-2-oxoindole, 2,3,4-trihydrothianaphthalene, or4-oxo-2,3-dihydrothianapthalene; and individual isomers, mixtures ofisomers, and pharmaceutically acceptable salts thereof provided that:when the multibinding compound of Formula (I) is a compound of formula:

 where Ar¹ and Ar³ are aryl, then W and X both are not alkylene oralkylene-O—.
 5. The bivalent multibinding compound of claim 4, wherein:X is —O—, —O-alkylene, —O-(arylene)-NH-(substituted alkylene),—O-(alkylene)-O-(arylene)-(alkylene)-O-(alkylene)-NH-(substitutedalkylene)-, —O-(alkylene)-O-(arylene)-, or-(alkylene)-(cycloalkylene)-NH-(substituted alkylene); and Q is acovalent bond.
 6. The multibinding compound of claim 5, wherein: Ar¹ is:(i) a phenyl ring of formula (c):

wherein: R⁴ is hydrogen, methyl, fluoro, chloro, or methoxy; R⁵ ishydrogen, hydroxy, fluoro, chloro, amino, or —NHSO₂CH₃; R⁶ is hydrogen,chloro, fluoro, hydroxy, methoxy, hydroxymethyl, —CH₂SO₂CH₃, —NHSO₂CH₃,—NHCHO, —CONH₂, or —NHCONH₂; Ar² is phenyl wherein the W and the Xgroups are attached at the 1, 4 position of the phenyl ring; and Ar³ iseither: (i) a phenyl ring of formula (c) as defined in claim 6 above; or(ii) a phenyl ring of formula (d):

wherein: R⁷ is hydrogen, methyl, propen-2-yl, fluoro, chloro, methoxy,—OCH₂CO₂Me, —OCON(NCH₃)₂, hydroxy, —CH₂CONH₂, —NHCOCH₃, imidazol-1-yl,or 1-methyl-4-trifluoromethyl-imidazol-2-yl; and R⁸ is hydrogen, fluoro,chloro, methoxy, —OCH₂CO₂Me, —OCON(CH₃)₂, or —CONH₂; or (iii) naphthyl,pyridyl, benzimidazol-1-yl, indolyl, 2-cyanoindolyl, carbazolyl,4-methylindanyl, 5-(CH₃CO₂CH₂O—)-1,2,3,4-tetrahydronaphthyl,1H-2-oxoindole, 2,3,4-trihydrothianaphthalene, or4-oxo-2,3-dihydrothianapthalene.
 7. The multibinding compound of claim 6wherein: Ar¹ is phenyl, 4-hydroxyphenyl, 3,4-dihydroxyphenyl,3,4-dichlorophenyl, 2-chloro-3,4-dihydroxyphenyl,2-fluoro-3,4-dihydroxyphenyl, 2-chloro-3,5-dihydroxyphenyl,2-fluoro-3,5-dihydroxyphenyl, 4-hydroxy-3-methoxyphenyl,4-hydroxy-3-hydroxymethylphenyl, 4-hydroxy-3-(HCONH—)phenyl,4-hydroxy-3-(NH₂CO—)phenyl, 3-chlorophenyl, 2,5-diethoxyphenyl,4-(CH₃SO₂NH—)-phenyl, 4-hydroxy-3-(CH₃SO₂CH₂—)phenyl,4-hydroxy-3-(CH₃SO₂NH—)phenyl, 4-hydroxy-3-(NH₂CONH—)phenyl, or3,5-dichloro-4-aminophenyl; W is a bond, methylene, ethylene, propylene,—(CH₂)₆—O—(CH₂)₃—, —(CH₂)₆—O—, or —CH₂CH(OH)CH₂—O—; X is —O—;—O—(CH₂)₄—; —O-(1,4-phenylene)-NH—CH₂—CH(OH)—;—O—(CH₂)₁₀—O-(1,4-phenylene)-(CH₂)₃—O—(CH₂)₆—NH—CH₂—CH(OH)—;—O-(CH₂)₆—O-(1,4-phenylene)-(CH₂)₃—O—(CH₂)₅—NH—CH₂—CH(OH)—;—O—(CH₂)₆—O-(1,4-phenylene)-; or —CH₂-(1,4-cyclohexyl)-NH—CH₂—CH(OH)—;and Ar³ is:


8. The multibinding compound of claim 4, wherein: X is a covalent bond;and Q is a substituted alkylene group wherein one or more of the carbonatoms in said substituted alkylene group is optionally replaced by aheteroatom selected from the group consisting of—NR^(a)— (where R^(a) ishydrogen, alkyl, or acyl) and —O—.
 9. The multibinding compound of claim8, wherein: Ar¹ is: (i) a phenyl ring of formula (c):

wherein: R⁴ is hydrogen, methyl, fluoro, chloro, or methoxy; R⁵ ishydrogen, hydroxy, fluoro, chloro, amino, or —NHSO₂CH₃; and R⁶ ishydrogen, chloro, fluoro, hydroxy, methoxy, hydroxymethyl, —CH₂SO₂CH₃,—NHSO₂CH₃, —NHCHO, —CONH₂, or —NHCONH₂; Ar² is phenyl wherein the W andthe X groups are attached at the 1, 4 position of the phenyl ring; andAr³ is either: (i) a phenyl ring of formula (c) as defined in claim 9above; or (ii) a phenyl ring of formula (d):

wherein: R⁷ is hydrogen, methyl, propen-2-yl, fluoro, chloro, methoxy,—OCH₂CO₂Me, —OCON(CH₃)₂, hydroxy, —CH₂CONH₂, —NHCOCH₃, imidazol-1-yl, or1-methyl-4-trifluoromethyl-imidazol-2-yl; and R⁸ is hydrogen, fluoro,chloro, methoxy, —OCH₂CO₂Me, —OCON(CH₃)₂, or —CONH₂; or (iii) naphthyl,pyridyl, benzimidazol-1-yl, indolyl, 2-cyanoindolyl, carbazolyl,4-methylindanyl, 5-(CH₃CO₂CH₂O—)-1,2,3,4-tetrahydronaphthyl,1H-2-oxoindole, 2,3,4-trihydrothianaphthalene, or4-oxo-2,3-dihydrothianapthalene.
 10. The multibinding compound of claim9 wherein: Ar¹ is phenyl, 4-hydroxyphenyl, 3,4-dihydroxyphenyl,3,4-dichlorophenyl, 2-chloro-3,4-dihydroxyphenyl,2-fluoro-3,4-dihydroxyphenyl, 2-chloro-3,5-dihydroxyphenyl,2-fluoro-3,5-dihydroxyphenyl, 4-hydroxy-3-methoxyphenyl,4-hydroxy-3-hydroxymethylphenyl, 4-hydroxy-3-(HCONH—)phenyl,4-hydroxy-3-(NH₂CO—)phenyl, 3-chlorophenyl, 2,5-dimethoxyphenyl,4-(CH₃SO₂NH—)-phenyl, 4-hydroxy-3-(CH₃SO₂CH₂—)phenyl,4-hydroxy-3-(CH₃SO₂NH—)phenyl, 4-hydroxy-3-(NH₂CONH—)phenyl, or3,5-dichloro-4-aminophenyl; W is a bond, methylene, ethylene, propylene,—(CH₂)₆—O—(CH₂)₃—, —(CH₂)₆—O—, or —CH₂CH(OH)CH₂—O—; Q is—NH—CH₂—CH(OH)—; —NH—CH(CH₂OH)—; —CH₂—NH—CH₂—CH(OH)—;—C(CH₃)₂—NH—CH₂—CH(OH)—; —(CH₂)₃—NH—CH₂—CH(OH)—;—(CH₂)₃—O—(CH_(2,6)—NH—CH₂—CH(OH)—; —(CH₂)₂—NH—CH₂ 13 CH(OH)—; or—O—(CH₂)—CH(OH)—CH₂—NH—CH₂—CH(OH)—; and Ar³ is:


11. The multibinding compound of claim 10 wherein the compound is:

where the stereochemistry at *C and **C is RS, R, or S.
 12. Themultibinding compound of claim 10 wherein the compound is:

where the stereochemistry at *C and **C is RS, R, or S.
 13. Themultibinding compound of claim 10 wherein the compound is:

where the stereochemistry at *C and **C is RS, R, or S.
 14. Themultibinding compound of claim 8 wherein: Ar³ is phenyl; and Q is—NH—CH₂—*CH(OH)-(where * is RS, R, or S stereochemistry).
 15. Themultibinding compound of claim 8 wherein: Ar³ is phenyl,4-hydroxy-3-(NH₂CONH—)phenyl, or 3,5-dichloro-4-aminophenyl.
 16. Themultibinding compound of claim 8 wherein: Ar¹ is phenyl,4-hydroxyphenyl, 3,4-dihydroxyphenyl, 3,4-dichlorophenyl,2-chloro-3,4-dihydroxyphenyl, 2-fluoro-3,4dihydroxyphenyl,2-chloro-3,5-dihydroxyphenyl, 2-fluoro-3,5-dihydroxyphenyl,4-hydroxy-3-methoxyphenyl, 4-hydroxy-3-hydroxymethylphenyl,4-hydroxy-3-(HCONH—)phenyl, 4-hydroxy-3-(NH₂CO—)phenyl, 3-chlorophenyl,2,5-dimethoxyphenyl, 4-(CH₃SO₂NH—)-phenyl,4-hydroxy-3-(CH₃SO₂CH₂—)phenyl, 4-hydroxy-3-(CH₃SO₂NH—)phenyl,4-hydroxy-3-(NH₂CONH—)phenyl, or 3,5-dichloro-4-aminophenyl; W is abond, methylene, ethylene, propylene, —(CH₂)₆—O—(CH₂)₃—, —(CH₂)₆—O—, or—CH₂CH(OH)CH₂—O—; Ar² is phenyl wherein the W and the X groups areattached at the 1,4-position of the phenyl ring; Q is—NH—CH₂—CH(OH)—CH₂—O—; and Ar³ is:


17. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and an effective amount of a multibinding compound ofFormula (I): (L)_(p)(X)_(q)  (I) wherein: p is an integer of from 2 to10; q is an integer of from 1 to 20; X is a linker; and L is a ligandwherein: one of the ligands, L, is a compound of formula (a):

wherein: Ar¹ l and Ar² are independently selected from the groupconsisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, andheterocyclyl wherein each of said Ar¹ and Ar² substituent optionallylinks the ligand to a linker; R¹ is selected from the group consistingof hydrogen, alkyl, and substituted alkyl, or R¹ is a covalent bondlinking the ligand to a linker; R² is selected from the group consistingof hydrogen, alkyl, aralkyl, acyl, substituted alkyl, cycloalkyl, andsubstituted cycloalkyl, or R² is a covalent bond linking the ligand to alinker; W is a covalent bond linking the —NR²— group to Ar², alkylene orsubstituted alkylene wherein one or more of the carbon atoms in saidalkylene or substituted alkylene group is optionally replaced by asubstituent selected from the group consisting of —NR^(a)— (where R^(a)is hydrogen, alkyl, acyl, or a covalent bond linking the ligand to alinker), —O—, —S(O)_(n) (where n is an integer of from 0 to 2), —CO—,—PR^(b)— (where R^(b) is alkyl), —P(O)₂—, and —O—P(O)O— and furtherwherein said alkylene or substituted alkylene group optionally links theligand to a linker provided that at least one of Ar¹, Ar², R¹, R², or Wlinks the ligand to a linker; and the other ligands are independently ofeach other a compound of formula (b): —Q—Ar³  (b) wherein: Ar³ isselected from the group consisting of aryl, heteroaryl, cycloalkyl,substituted cycloalkyl, and heterocyclyl; Q, which links the otherligand to the linker, is selected from the group consisting of acovalent bond, alkylene, and a substituted alkylene group wherein one ormore of the carbon atoms in said alkylene and substituted alkylene groupis optionally replaced by a substituent selected from the groupconsisting of —NR^(a)— (where R^(a) is hydrogen, alkyl, acyl, or acovalent bond linking the ligand to a linker), —O—, —S(O)_(n)— (where nis an integer of from 0 to 2), —CO—, —PR^(b)— (where R^(b) is alkyl),—P(O)₂—, and —O—P(O)O—; and individual isomers, mixtures of isomers andpharmaceutically acceptable salts thereof provided that: (i) when themultibinding compound of Formula (I) is a compound of formula:

where Ar¹ and Ar³ are aryl, then W and X both are not alkylene oralkylene-O—; (ii) when the multibinding compound of Formula (I) is acompound of formula:

where Ar¹ is 4-hydroxy-2-methylphenyl, Ar² is aryl, Ar³ is aryl orheterocyclyl, W is ethylene, Q is a covalent bond, R¹ is alkyl, then thelinker X is not linked to the Ar² group through an oxygen atom; (iii)when the multibinding compound of Formula (I) is a compound of formula:

where Ar¹, Ar², Ar^(3,) R¹, R² are as defined above, W is alkylene, andQ is a covalent bond, then X is not -alkylene-O—; and (iv) when themultibinding compound of Formula (I) is a compound of formula:

where Ar¹is 4-benzyloxy-3-formylamino, R² is aralkyl, W is —CH(CH₃)CH₂—,Ar² and Ar³ are phenyl, Q is a covalent bond, then the linker X is notlinked to the Ar² group through an oxygen atom.
 18. The pharmaceuticalcomposition of claim 17 wherein q is less than p.
 19. The pharmaceuticalcomposition of claim 18 wherein each linker independently has theformula: —X^(a)—Z—(Y^(a)—Z)_(m)—X^(a)— wherein m is an integer of from 0to 20; X^(a) at each separate occurrence is selected from the groupconsisting of —O—, —S—, —NR—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)NR—,—NRC(O)—, C(S), —C(S)O—, —C(S)NR—, —NRC(S)—, or a covalent bond where Ris as defined below; Z at each separate occurrence is selected from thegroup consisting of alkylene, substituted alkylene, cycloalkylene,substituted cycloalkylene, alkenylene, substituted alkenylene,alkynylene, substituted alkynylene, cycloalkenylene, substitutedcycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalentbond; each Y^(a) at each separate occurrence is selected from the groupconsisting of —O—, —C(O)—, —OC(O)—, —C(O)O—, —NR—, —S(O)_(n)—,—C(O)NR′—, —NR′C(O)—, —NR′C(O)NR′—, —NR′C(S)NR′—, —C(═NR′)—NR′—,—NR′—C(═NR′)—, —OC(O)—NR′—, —NR′—C(O)—O—, —N═C(X^(a))—NR′—,—NR′—C(X^(a))═N—, —P(O)(OR′)—O—, —O—P(O)(OR′)—, —S(O)_(n)CR′R″—,—S(O)_(n)—NR′—, —NR′—S(O)_(n)—, —S—S—, and a covalent bond; where n is0, 1 or 2; R, R′ and R″ at each separate occurrence are selected fromthe group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl,substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryland heterocyclic, and X^(a) is as defined above.
 20. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and aneffective amount of a multibinding compound of claim
 7. 21. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and an effective amount of a multibinding compound of claim 10.22. A method for treating diseases mediated by a β2 adrenergic receptorin a mammal, said method comprising administering to said mammal atherapeutically effective amount of a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a multibindingcompound of Formula (I): (L)_(p)(X)_(q)  (I) wherein: p is an integer offrom 2 to 10; q is an integer of from 1 to 20; X is a linker, and L is aligand wherein: one of the ligands, L, is a compound of formula (a):

wherein: Ar¹ and Ar² are independently selected from the groupconsisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, andheterocyclyl wherein each of said Ar¹ and Ar² substituent optionallylinks the ligand to a linker; R¹ is selected from the group consistingof hydrogen, alkyl, and substituted alkyl, or R¹ is a covalent bondlinking the ligand to a linker; R² is selected from the group consistingof hydrogen, alkyl, aralkyl, acyl, substituted alkyl, cycloalkyl, andsubstituted cycloalkyl, or R² is a covalent bond linking the ligand to alinker; W is a covalent bond linking the —NR²— group to Ar², alkylene orsubstituted alkylene wherein one or more of the carbon atoms in saidalkylene or substituted alkylene group is optionally replaced by asubstituent selected from the group consisting of —NR^(a)— (where R^(a)is hydrogen, alkyl, acyl, or a covalent bond linking the ligand to alinker), —O—, —S(O)_(n) (where n is an integer of from 0 to 2), —CO—,—PR^(b)— (where R^(b) is alkyl), —P(O)₂—, and —O—P(O)O— and furtherwherein said alkylene or substituted alkylene group optionally links theligand to a linker provided that at least one of Ar¹, Ar², R¹, R², or Wlinks the ligand to a linker; and the other ligands are independently ofeach other a compound of formula (b): —Q—Ar³  (b) wherein: Ar³ isselected from the group consisting of aryl, heteroaryl, cycloalkyl,substituted cycloalkyl, and heterocyclyl; Q, which links the otherligand to the linker, is selected from the group consisting of acovalent bond, alkylene, and substituted alkylene wherein one or more ofthe carbon atoms in said alkylene or substituted alkylene is optionallyreplaced by a substituent selected from the group consisting of —NR^(a)—(where R^(a) is hydrogen, alkyl, acyl, or a covalent bond linking theligand to a linker), —O—, —S(O)_(n)— (where n is an integer of from 0 to2), —CO—, —PR^(b)— (where R^(b) is alkyl), —P(O)₂—, and —O—P(O)O—; andindividual isomers, mixtures of isomers and pharmaceutically acceptablesalts thereof provided that: (i) when the multibinding compound ofFormula (I) is a compound of formula:

 where Ar¹ and Ar³ are aryl, then W and X both are not alkylene oralkylene-O—; (ii) when the multibinding compound of Formula (I) is acompound of formula:

 where Ar¹ is 4-hydroxy-2-methylphenyl, Ar² is aryl, Ar³ is aryl orheterocyclyl, W is ethylene, Q is a covalent bond, R¹ is alkyl, then thelinker X is not linked to the Ar² group through an oxygen atom; (iii)when the multibinding compound of Formula (I) is a compound of formula:

 where Ar¹, Ar², Ar³, R¹, R² are as defined above, W is alkylene, and Qis a covalent bond, then X is not -alkylene-O—; and (iv) when themultibinding compound of Formula (I) is a compound of formula:

 where Ar¹ is 4-benzyloxy-3-formylamino, R² is aralkyl, W is—CH(CH₃)CH₂—, Ar² and Ar³ are phenyl, Q is a covalent bond, then thelinker X is not linked to the Ar² group through an oxygen atom.
 23. Amethod for treating diseases mediated by a β2 adrenergic receptor in amammal, said method comprising administering to said mammal atherapeutically effective amount of a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a multibindingcompound of claim
 7. 24. A method for treating diseases mediated by a β2adrenergic receptor in a mammal, said method comprising administering tosaid mammal a therapeutically effective amount of a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and amultibinding compound of claim
 10. 25. The method of claim 24 whereinthe disease is a respiratory disease.
 26. The method of claim 25 whereinthe disease is asthma or chronic pulmonary obstructive disease.
 27. Amethod for identifying multimeric ligand compounds possessingmultibinding properties for β2 adrenergic receptor which methodcomprises: (a) identifying a ligand or a mixture of ligands wherein eachligand contains at least one reactive functionality; (b) identifying alibrary of linkers wherein each linker in said library comprises atleast two functional groups having complementary reactivity to at leastone of the reactive functional groups of the ligand; (c) preparing amultimeric ligand compound library by combining at least twostoichiometric equivalents of the ligand or mixture of ligandsidentified in (a) with the library of linkers identified in (b) underconditions wherein the complementary functional groups react to form acovalent linkage between said linker and at least two of said ligands;and (d) assaying the multimeric ligand compounds produced in the libraryprepared in (c) above to identify multimeric ligand compounds possessingmultibinding properties for β2 adrenergic receptor.
 28. A method foridentifying multimeric ligand compounds possessing multibindingproperties for β2 adrenergic receptor which method comprises: (a)identifying a library of ligands wherein each ligand contains at leastone reactive functionality; (b) identifying a linker or mixture oflinkers wherein each linker comprises at least two functional groupshaving complementary reactivity to at least one of the reactivefunctional groups of the ligand; (c) preparing a multimeric ligandcompound library by combining at least two stoichiometric equivalents ofthe library of ligands identified in (a) with the linker or mixture oflinkers identified in (b) under conditions wherein the complementaryfunctional groups react to form a covalent linkage between said linkerand at least two of said ligands; and (d) assaying the multimeric ligandcompounds produced in the library prepared in (c) above to identifymultimeric ligand compounds possessing multibinding properties for β2adrenergic receptor.
 29. The method according to claim 27 or 28 whereinthe preparation of the multimeric ligand compound library is achieved byeither the sequential or concurrent combination of the two or morestoichiometric equivalents of the ligands identified in (a) with thelinkers identified in (b).
 30. The method according to claim 29 whereinthe multimeric ligand compounds comprising the multimeric ligandcompound library are dimeric.
 31. The method according to claim 30wherein the dimeric ligand compounds comprising the dimeric ligandcompound library are heterodimeric.
 32. The method according to claim 31wherein the heterodimeric ligand compound library is prepared bysequential addition of a first and second ligand.
 33. The methodaccording to claim 27 or 28 wherein, prior to procedure (d), each memberof the multimeric ligand compound library is isolated from the library.34. The method according to claim 33 wherein each member of the libraryis isolated by preparative liquid chromatography mass spectrometry(LCMS).
 35. The method according to claim 27 or claim 28 wherein thelinker or linkers employed are selected from the group comprisingflexible linkers, rigid linkers, hydrophobic linkers, hydrophiliclinkers, linkers of different geometry, acidic linkers, basic linkers,linkers of different polarization and amphiphilic linkers.
 36. Themethod according to claim 35 wherein the linkers comprise linkers ofdifferent chain length and/or having different complementary reactivegroups.
 37. The method according to claim 36 wherein the linkers areselected to have different linker lengths ranging from about 2 to 100 Å.38. The method according to claim 27 or 28 wherein the ligand or mixtureof ligands is selected to have reactive functionality at different siteson said ligands.
 39. The method according to claim 38 wherein saidreactive functionality is selected from the group consisting ofcarboxylic acids, carboxylic acid halides, carboxyl esters, amines,halides, pseudohalides, isocyanates, vinyl unsaturation, ketones,aldehydes, thiols, alcohols, anhydrides, boronates, and precursorsthereof wherein the reactive functionality on the ligand is selected tobe complementary to at least one of the reactive groups on the linker sothat a covalent linkage can be formed between the linker and the ligand.40. The method according to claim 27 or claim 28 wherein the multimericligand compound library comprises homomeric ligand compounds.
 41. Themethod according to claim 27 or claim 28 wherein the multimeric ligandcompound library comprises heteromeric ligand compounds.
 42. A libraryof multimeric ligand compounds which may possess multivalent propertiesfor β2 adrenergic receptor which library is prepared by the methodcomprising: (a) identifying a ligand or a mixture of ligands whereineach ligand contains at least one reactive functionality; (b)identifying a library of linkers wherein each linker in said librarycomprises at least two functional groups having complementary reactivityto at least one of the reactive functional groups of the ligand; and (c)preparing a multimeric ligand compound library by combining at least twostoichiometric equivalents of the ligand or mixture of ligandsidentified in (a) with the library of linkers identified in (b) underconditions wherein the complementary functional groups react to form acovalent linkage between said linker and at least two of said ligands.43. A library of multimeric ligand compounds which may possessmultivalent properties for β2 adrenergic receptor which library isprepared by the method comprising: (a) identifying a library of ligandswherein each ligand contains at least one reactive functionality; (b)identifying a linker or mixture of linkers wherein each linker comprisesat least two functional groups having complementary reactivity to atleast one of the reactive functional groups of the ligand; and (c)preparing a multimeric ligand compound library by combining at least twostoichiometric equivalents of the library of ligands identified in (a)with the linker or mixture of linkers identified in (b) under conditionswherein the complementary functional groups react to form a covalentlinkage between said linker and at least two of said ligands.
 44. Thelibrary according to claim 42 or claim 43 wherein the linker or linkersemployed are selected from the group comprising flexible linkers, rigidlinkers, hydrophobic linkers, hydrophilic linkers, linkers of differentgeometry, acidic linkers, basic linkers, linkers of differentpolarization and/or polarizability, and amphiphilic linkers.
 45. Thelibrary according to claim 44 wherein the linkers comprise linkers ofdifferent chain length and/or having different complementary reactivegroups.
 46. The library according to claim 45 wherein the linkers areselected to have different linker lengths ranging from about 2 to 100 Å.47. The library according to claim 42 or 43 wherein the ligand ormixture of ligands is selected to have reactive functionality atdifferent sites on said ligands.
 48. The library according to claim 47wherein said reactive functionality is selected from the groupconsisting of carboxylic acids, carboxylic acid halides, carboxylesters, amines, halides, pseudohalides, isocyanates, vinyl unsaturation,ketones, aldehydes, thiols, alcohols, anhydrides, boronates, andprecursors thereof wherein the reactive functionality on the ligand isselected to be complementary to at least one of the reactive groups onthe linker so that a covalent linkage can be formed between the linkerand the ligand.
 49. The library according to claim 42 or claim 43wherein the multimeric ligand compound library comprises homomericligand compounds.
 50. The library according to claim 42 or claim 43wherein the multimeric ligand compound library comprises heteromericligand compounds.
 51. An iterative method for identifying multimericligand compounds possessing multibinding properties for β2 adrenergicreceptor which method comprises: (a) preparing a first collection oriteration of multimeric compounds which is prepared by contacting atleast two stoichiometric equivalents of the ligand or mixture of ligandswhich target a receptor with a linker or mixture of linkers wherein saidligand or mixture of ligands comprises at least one reactivefunctionality and said linker or mixture of linkers comprises at leasttwo functional groups having complementary reactivity to at least one ofthe reactive functional groups of the ligand wherein said contacting isconducted under conditions wherein the complementary functional groupsreact to form a covalent linkage between said linker and at least two ofsaid ligands; (b) assaying said first collection or iteration ofmultimeric compounds to assess which if any of said multimeric compoundspossess multibinding properties for β2 adrenergic receptor; (c)repeating the process of (a) and (b) above until at least one multimericcompound is found to possess multibinding properties for β2 adrenergicreceptor; (d) evaluating what molecular constraints impartedmultibinding properties to the multimeric compound or compounds found inthe first iteration recited in (a)-(c) above; (e) creating a secondcollection or iteration of multimeric compounds which elaborates uponthe particular molecular constraints imparting multibinding propertiesfor β2 adrenergic receptor to the multimeric compound or compounds foundin said first iteration; (f) evaluating what molecular constraintsimparted enhanced multibinding properties to the multimeric compound orcompounds found in the second collection or iteration recited in (e)above; (g) optionally repeating steps (e) and (f) to further elaborateupon said molecular constraints.
 52. The method according to claim 51wherein steps (e) and (f) are repeated from 2-50 times.
 53. The methodaccording to claim 52 wherein steps (e) and (f) are repeated from 5-50times.